Containers and kits for the determination of cell functions, and method for the determination thereof

ABSTRACT

A container for measurement of cell functions, for use in the determination of a physiologically active substance produced by blood cells, is constituted such that an amount of material capable of inducing production of the physiologically active substance, when extracted by collecting water of a volume equal to a liquid volume to be subjected to measurement, is controlled at a level insufficient to induce production of the physiologically active substance from the blood cells. A container for measurement of cell functions in which a material capable of inducing production of a physiologically active substance in blood when contacted with the blood is accommodated in such a condition as being contactable with blood, and in which an amount of the material capable of inducing production of the physiologically active substance in the container before use is limited to a level insufficient to influence a measured value of the physiologically active substance.

FIELD OF THE INVENTION

The present invention relates to a container for measurement of cellfunctions involved in immune and inflammatory reactions, a kit formeasurement of the cell functions and a method for measuring the cellfunctions, and more particularly to a container for measurement of cellfunctions which is carried out through determination of physiologicallyactive substances, such as cytokines, produced by blood cells includinggranulocytes, monocytes, macrophages, lymphocytes or the like, forexample, kit and method for such a measurement of cell functions.

DESCRIPTION OF PRIOR ART

Leukocytes, such as granulocytes, monocytes, macrophages andlymphocytes, play various rolls in diversified bioprotective reactionsincluding immune and inflammatory reactions in blood or respectiveorgans. It is known that these cells exhibit important functions in avariety of morbidities including infectious diseases; inflammatorydiseases such as hepatitis and nephritis; immune.allergic diseases suchas rheumatoid arthritis and asthma; and cancer, and the functions ofthese cells are either suppressed or enhanced as the morbidity varies.

It is also known that a variety of drugs, such as anti-inflammatorydrugs, immunosuppressants, immunoenhancers and anticancer drugs, areuseful in the therapy of these diseases, wherein the functions of thesecells are either suppressed or enhanced concurrently. It is thereforeimportant to examine the functions of these cells whereby themorbidities of various diseases, effects and side-effects of drugs canbe identified to determine therapeutic schemes, doses of the drugs andtimings of the drug administration.

In view of the above-described reasons, a granulocyte phagocyticactivity test, a granulocyte bactericidal activity (active oxygenproducing capacity) test, a lymphocyte transformation test and the likehave been conventionally conducted at hospital examining rooms orcenters in order to measure such cell functions. Also in recent years, asurface antigen test has been carried out which utilizes a flowcytometer and fluorescence-labelled monoclonal antibodies againstrespective surface antigens of various immunocompetent cells. However,the conventional testing methods have required such specializedtechniques as separation and culture of cells, microscopic measurementor the like, to consequently necessitate time-consuming measurements, RIfacilities and expensive equipments.

Also, monocytes in blood, as well as macrophages into which themonocytes moved into tissues differentiates and matures, have a widespectrum of functions, for example, as coming into play in foreign bodyexclusion through phagocytosis and immune formation through antigenpresentation, or as secreting various physiologically active substances,such as cytokine and prostaglandin, to thereby regulate an inflammatoryor immune reaction. Like granulocytes and lymphocytes, these monocytesand macrophages play important rolls also in a variety of morbidities.It is therefore very important to identify the functions of these cells.Particularly in infectious diseases, unlike the granulocytes andlymphocytes, the monocytes and macrophages exhibit slight changes interms of the number of cells and primarily amplify their functions, sothat the measurement of changes in cell functions becomes more important(“Macrophages”, written by Tohru Tokunaga, Kodansha Scientific, 1st Ed.published in 1986).

Tumor necrosis factor α (hereinafter referred to as TNFα),interleukin-1β (hereinafter referred to as IL-1β), and interleukin-6(hereinafter referred to as IL-6), all called as monokine, are cytokineswhich are produced mainly by leukocytes including monocytes andmacrophages, among blood cells, and which come into play in variousinflammatory and immune reactions.

A variety of methods reported to date examines the above-describedcytokine-producing functions of blood or leukocytes separated fromblood. For example, in gazettes of Patent Laying-open Nos. Hei 2-196961and Hei 3-285692, methods are disclosed which react lipopolysaccharide(LPS) or lectin with blood to induce production of cytokines, such asTNFα or IL-1β, which are subsequently quantitatively determined. Also,in gazettes of Patent Laying-open Nos. Hei 6-209992 and Hei 7-67955,methods are disclosed which react blood with polymer materials having aspecific surface roughness or chemical structures to induce productionof TNFα. Also, in gazettes of Patent Laying-open Nos. Hei 7-299732 andHei 7-151752, bioreaction tests are disclosed which react blood withpolymer materials having a specific surface roughness to determine theamount of produced TNFα or IL-1β. Also, in a gazette of Tokkohyo No. Hei7-500905, a method is disclosed which measures immunoactivity of atested substance by determining the production of cytokines, such asTNFα or IL-1β, induced from human peripheral blood leukocytes.

However, the above-described methods for measurement of cell functionswhich have been conventionally carried out at hospital examining roomsor centers, as well as the methods disclosed in the gazettes of theabove-listed laid-open patents, have the following problems. That is,these tests all require specialized operations, such as an operation ofcollecting blood from an examined human using an injector and thereaftermanually transferring the blood to various reactors as by pipetting,cell separation for separating leukocytes and the others, cell culturefor the purpose of measuring cell functions or the like. This carries arisk for an examining person to acquire various infectious diseases,such as hepatitis and AIDS, when the person contacts the blood. Also,there is a possibility that various bacteria or dusts are accidentallyincorporated into a specimen blood during such operations. There existsanother risk of adversely affecting the measurement results when thosecontaminants or operations physically stimulate the cells in bloodwithout necessity.

Particularly in the conventional methods which employ a specific reactorto collect blood therein and determine the production of cytokines,specifically of TNFα or IL-1β induced from leukocytes, there has existedan occasion that the endotoxin, such as LPS derived from gram-negativebacteria, have been originally incorporated in a blood collectingequipment, such as an injector, or in a reactor. Since even a veryslight amount of endotoxin can induce production of TNFα or IL-1β fromleukocytes, it was impossible to obtain reliable measurement resultswhen, for example, entry of a slight amount of dusts during amanufacturing process or contamination through employed cleaning waterresulted in incorporation of a small amount of endotoxin in theabove-described blood collecting equipment or reactor.

In view of the above-described problems, a method for measurement ofcell functions is sought which is more simplified in its operations,less risky and more accurate than conventional methods.

In another aspect, anticoagulants have been conventionally utilized whenmeasuring various physiologically active substances in blood, functionsof blood cells, surface antigens of blood cells or the like.

However, there exists no general standard for endotoxin contents inanticoagulant, other than a guideline given by “Endotoxin testingmethod” in the dispensatory of 13th revised Japanese Pharmacopeia,which, for anticoagulants employed as an injection drug, officially sets5 EU/Kg as a standard for a specification of minimal pyrogenic dose to arabbit.

Endotoxin is lipopolysaccharide constituting a cell-wall outer membraneof gram-negative bacteria, and a very slight amount thereof suffices tostimulate blood cells, such as leukocytes, to produce physiologicallyactive substances such as a variety of cytokines including TNFα, IL-1β,IL-6, or granulocyte-macrophage colony-stimulating factors. They exhibitvarious physiological actions such as pyrogenic activity and endotoxinshock (Nippon Igaku-kan “Inflammation and cytokines '87 InflammationSeminar”, p.103-108).

Also, the physiologically active substances, such as the above-describedcytokines produced from blood cells, interact with each other inautocrine or paracrine mode to cause further production of histamines,arachidonate metabolites or various cytokines, to modify variousfunctions of blood cells, and to cause quantitative and qualitativechanges of blood cell surface antigens.

Accordingly, if anticoagulants are contaminated with endotoxin, and ifthe contents of such endotoxin are in a sufficient level to causeproduction of the physiologically active substances, it would becomeimpossible to carry out precise measurements of various physiologicallyactive substances in blood, of functions of blood cells, and of surfaceantigens of blood cells.

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide a container, as wellas a kit and a method, respectively for measurement of cell functions,which can eliminate the problems imposed upon the conventional methodsfor measurement of cell functions, which is more simplified inoperations and less risky than conventional, and which can measure cellfunctions at an increased accuracy than conventional.

In accordance with a broad aspect of a first invention, in order toaccomplish the above-described object, a container for measurement ofcell functions, for use in determining physiologically active substancesproduced from blood cells, is provided which is characterized in that anamount of material capable of inducing production of the above-describedphysiologically active substances, when extracted by collecting water ofa volume equal to a liquid volume to be subjected to measurement, is ata level insufficient to induce production of the physiologically activesubstances from the blood cells.

Since, in this container for measurement of cell functions, as describedabove, the amount of material capable of inducing production of thephysiologically active substances, when extracted by collecting water ofa volume equal to a liquid volume to be subjected to measurement, islimited to a level insufficient to induce production of thephysiologically active substances from the blood cells, that is, thecontainer for measurement of cell functions contains in itself theabove-specified limited amount of the material capable of inducingproduction of the physiologically active substances, the collected bloodis scarcely subjected to unnecessary stimulation for a period fromcollection till measurement so that a long-term preservation thereof isenabled. This allows precise measurement of the physiologically activesubstances present in the collected blood and enables the use of thecontainer in precisely examining morbidities of patients having variousdiseases.

Also, the container for measurement of cell functions in accordance withthis first invention can be suitably employed to obtain control values,when used in combination with a container for measurement of cellfunctions in accordance with a below-described second invention.

In the container for measurement of cell functions in accordance withthe first invention of the present application, the material capable ofinducing production of the above-described physiologically activesubstances is preferably endotoxin, and its content in the container formeasurement of cell functions before use is specified not to exceed 0.5EU/ml as a concentration in an extracted liquid when extracted bycollecting water of a volume equal to a liquid volume to be subjected tomeasurement.

The second invention of the present application is a container formeasurement of cell functions which is characterized in that a materialwhich induces production of physiologically active substances in bloodupon contact with the blood, is accommodated therein in such a conditionas being contactable with blood, and that the amount, present in thecontainer before use, of the above-described material capable ofinducing production of physiologically active substances is limited soas not to adversely affect measured values of the physiologically activesubstances as described above.

In the container for measurement of cell functions in accordance withthe second invention, although the material capable of inducingproduction of the physiologically active substances in blood isaccommodated in such a condition as being contactable with blood, thecontent of the material capable of inducing production of thephysiologically active substances, originally present in the containerbefore accommodation thereof, is limited, as described above, so as notto influence measured values of the physiologically active substances.Accordingly, the production of physiologically active substances can bedetermined very accurately when the blood is introduced and contactedwith the material capable of inducing production of the physiologicallyactive substances to thereby produce the physiologically activesubstances.

In the container for measurement of cell functions in accordance withthe second invention, the material which induces production of theabove-described physiologically active substances is preferablyendotoxin, and a concentration of endotoxin in a resulting whole liquidwhen contacted with blood is limited as being in the range of 0.6-100000EU/ml.

Also, in the container for measurement of cell functions in accordancewith the first or second invention, anticoagulants may be furtherincorporated therein to prevent blood coagulation.

Also, the amount of the material capable of inducing production ofphysiologically active substances contained in the above-describedanticoagulant is preferably at a level insufficient to induce productionof the physiologically active substances from blood cells when mixedwith blood.

In a particular aspect of the first and second inventions of the presentapplication, the material capable of inducing production ofphysiologically active substances is endotoxin while the physiologicallyactive substances are cytokines.

At least one species selected from tumor necrosis factor α (TNFα),interleukin-1β (IL-1β) and interleukin-6 (IL-6) can be cited as theabove-described cytokines.

Also, an interior of the container for measurement of cell functions, inaccordance with the first and second inventions, is preferably vacuumed.

Also, the containers for measurement of cell functions, in accordancewith the first and second inventions, can be combined with a reagentcapable of quantitating the induced physiologically active substances tothereby constitute a kit for measurement of cell functions. In such anevent, an enzyme immunoassay reagent, for example, can be employed asthe reagent capable of quantitating the induced physiologically activesubstances.

A third invention of the present application is a kit for measurement ofcell functions. The kit has a first container for measurement of cellfunctions in which an amount of a material capable of inducingproduction of the above-described physiologically active substances,when extracted by collecting water which does not contain the materialcapable of inducing production of physiologically active substances andhas such a volume as equal to a liquid volume to be subjected tomeasurement, is rendered at a level insufficient to induce production ofthe physiologically active substances from blood cells, and in whichanticoagulant may be contained when needed; a second container formeasurement of cell functions in which a material capable of inducingproduction of physiologically active substances in blood when contactedwith the blood, as well as anticoagulant, are accommodated in such acondition as to be contactable with blood, and in which the amount,originally present in the container before accommodation thereof, of theabove-described material capable of inducing production ofphysiologically active substances is limited so as not to adverselyaffect measured values of the physiologically active substances asdescribed above; and a reagent for quantitatively determining thephysiologically active substances. That is, the container formeasurement of cell functions according to the third invention has aconstitution combining the container for measurement of cell functionsaccording to the first invention, the container for measurement of cellfunctions according to the second invention, and the above-definedreagent for quantification.

In the kit for measurement of cell functions according to the thirdinvention, the above-specified reagent for quantification preferablyincludes a first enzyme immunoassay reagent for use in determination ofthe amount of physiologically active substances in blood collected inthe first container for measurement of cell functions; and a secondenzyme immunoassay reagent which is employed to determine the amount ofphysiologically active substances produced through a reaction of thematerial capable of inducing production of the physiologically activesubstances with the blood collected in the second container formeasurement of cell functions, and which is different in sensitivity tothe physiologically active substances from the first enzyme immunoassayreagent.

Also, in a particular aspect of the kit for measurement of cellfunctions according to the third invention, the above-described materialwhich induces production of the physiologically active substances isendotoxin, and a concentration of endotoxin in a resulting whole liquidwhen contacted with blood is rendered in the range of 0.6-100000 EU/ml.

A fourth invention of the present application is a method formeasurement of cell functions which characteristically includes a stepof introducing blood into the container according to the secondinvention for measurement of cell functions and reacting the introducedblood with a material capable of inducing production of physiologicallyactive substances to thereby induce production of the physiologicallyactive substances. In the method for measurement of cell functionsaccording to the fourth invention, the production of above-describedphysiologically active substances is preferably induced at a temperatureof 26-45° C.

Also, a preferred time period during which the production ofphysiologically active substances is induced is 1-6 hours. Furthermore,in the method for measurement of cell functions according to the fourthinvention, the amount of physiologically active substances is determinedby reagents capable of quantitating thereof. Also, in a more particularaspect of the method for measurement of cell functions according to thefourth invention, blood is introduced into the first and secondcontainers for measurement of cell functions in the kit for measurementof cell functions to induce production of the physiologically activesubstances. Again in this case, the production of physiologically activesubstances is preferably induced at a temperature of 26-45° C., and apreferred time period during which the production of physiologicallyactive substances is induced is 1-6 hours.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be now explained in detail.

(Container for Measurement of Cell Functions according to The FirstInvention)

For the container for measurement of cell functions according to thefirst invention, the amount of material capable of inducing productionof the above-described physiologically active substances, when extractedby collecting water devoid of the material capable of inducingproduction of physiologically active substances and of a volume equal toa liquid volume to be subjected to measurement, is required to be at alevel insufficient to induce production of the physiologically activesubstances from the blood cells.

The extraction method is actually carried out by collecting, into theabove container for measurement of cell functions, water devoid of thematerial capable of inducing production of physiologically activesubstances and of a volume equal to a liquid volume to be subjected tomeasurement, and by extracting under agitation for one hour at 37° C.

The above physiologically active substances are preferably cytokines andthe material capable of inducing production of physiologically activesubstances is preferably endotoxin. However, the physiologically activesubstances are not limited to cytokines, and can be arachidonatemetabolites such as prostaglandin, active oxygen species, solubleadhesion factors, soluble receptors, or intragranular enzymes, forexample. The type of material capable of inducing production ofphysiologically active substances can be suitably selected dependingupon the type of the physiologically active substances.

In such a case where the material capable of inducing production ofphysiologically active substances is endotoxin, the endotoxin content inan extracted solution resulting from the above-described extraction isspecified not to exceed 0.5 EU/ml (international endotoxin unit). If theabove endotoxin content exceeds 0.5 EU/ml, endotoxin is likely to causea marked induction of cytokines, one class of the physiologically activesubstances, in the collected blood, and the induced cytokines possiblystimulate various immunocompetent cells to cause changes in functionsthereof, consequently making precise measurements of cell functionsimpossible.

The liquid volume to be subjected to measurement, as described above,refers to a total volume of liquids employed when below-describedvarious measurements are carried out, e.g. a sum of a volume of blood tobe measured, a volume of a below-described anticoagulant solution addedwhen needed, a volume of a blood coagulation enhancer solution, and avolume of liquid for dissolving or suspending stimulators.

It should be understood here that the amount in volume of theendotoxin-free water when used in effecting the above-describedextraction is not required as being exactly equal to the liquid volumeto be subjected to measurement, and it may be below the liquid volume tobe subjected to measurement so far as the extraction can be carried outsuccessfully. Even in such a case, the function and effect of thepresent invention can be obtained, provided that the endotoxin contentin the extracted solution does not exceed 0.5 EU/ml.

Although there exists a variety of methods for determining the aboveendotoxin content, a method referenced in the present specification fordetermining the endotoxin content is a colorimetry, according to“Endotoxin testing method” in the dispensatory of 13th revised JapanesePharmacopeia. The colorimetry uses, as an indication, a color producedin a chromophoric synthetic substrate when hydrolyzed, and can becarried out, for example, using a commercially available productENDOSPECIE (manufactured by Seikagaku Kogyo Co.).

As a method for removing or deactivating endotoxin, various well-knowntechniques may be employed which include deactivation through heating,acid or alkali treatment; ultrafiltration using a membrane filter; andremoval using anionic chitosan resins, polymyxin B specifically bindableto endotoxin, or adsorbents which fixes antibodies against endotoxin.Also, instruments and containers employed for the endotoxin removingoperations are subjected to dry heat treatment at 250° C. for an hour orlonger, if they are made of glass, or immersed in a 0.2 M aqueoussolution of sodium hydroxide and cleaned with endotoxin-free water, ifthey are made of plastics, to insure complete deactivation of endotoxinprior to use thereof. Also, the endotoxin-free water must beconsistently used whenever water is needed, and an operating atmosphereis preferably such an atmosphere that any secondary endotoxincontamination is constrained within a practicable range, as provided bya clean room.

An interior of the container for measurement of cell functions,according to the first invention, is preferably vacuumed. The interiorpressure may be reduced to such an extent that atmospheric blood can besuctioned into the container for measurement of cell functions uponcommunication thereof with the interior of container for measurement ofcell functions. The interior pressure can be determined depending upon ablood amount to be suctioned. That is, the larger a target amount ofblood to be suctioned is, the higher degree of pressure reduction may beeffected.

The shape of container for measurement of cell functions, according tothe first invention, is not particularly specified, and may be tubularas exemplified by blood collecting tubes, test tubes and the like, orplate-like as represented by microplates. Preferably, it is suited forvacuum operation.

Illustrative of the type of material for the container for measurementof cell functions are glasses or plastics. Thermoplastic andthermosetting resins can be both employed as the above-describedplastics. Cited as examples of the thermoplastic resins arepolyethylene, polypropylene, polystyrene, polymethyl methacrylate,polyvinyl chloride, polyethylene terephthalate, styrene-acrylonitrilecopolymer, styrene-maleic anhydride copolymer, styrene-acrylatecopolymer, styrene-methylmethacrylate copolymer, ethylene-propylenecopolymer, ethylene-acrylic acid copolymer, and ethylene-acrylatecopolymer. Cited as examples of the thermosetting resins are unsaturatedpolyester resins, epoxy resins, and epoxy-acrylate resins.

In the event that a tubular part is employed as the container formeasurement of cell functions, according to the first invention, astopper is generally used to maintain the interior of the container at areduced pressure. Cited as exemplary material types for the stopper arebutyl rubbers, chlorinated butyl rubbers, thermoplastic elastomers andthe like.

If the tubular part is employed as the container for measurement of cellfunctions, according to the first invention, the amount of blood to becollected is varied depending upon a volume of the container formeasurement of cell functions, but may be generally about 0.5-2 ml incase of using a container having a volume of 4-5 ml.

On the other hand, if a microplate-like part is employed as thecontainer for measurement of cell functions, according to the firstinvention, the amount of blood to be collected may be about 0.05-2 ml.

If necessary, anticoagulant may be accommodated in the container formeasurement of cell functions, according to the first invention, toprevent blood coagulation. The above-described anticoagulant may bepresent in either liquid or solid form in the container. Cited as theabove anticoagulants are heparin compounds, citric acid compounds,oxalic acid compounds and the like. Heparin sodium is more preferablesince it does not inhibit biological reactions of cells. When blood iscollected in the container, a reduced concentration of heparin sodium inblood possibly causes blood coagulation while an increased concentrationthereof is likely to cause unexpected activation or inactivation ofcells. Accordingly, a preferred amount of heparin sodium accommodated inthe container is 4-50 U/ml.

An exemplary case where the anticoagulant is necessary to be added intothe container for measurement of cell functions, according to the firstinvention, is the case where determination is made as to physiologicallyactive substances produced (released or induced) by reacting cells, suchas monocytes, macrophages, lymphocytes, leukocytes or the like, withmaterial capable of inducing production of the physiologically activesubstances. Also, an exemplary case in which the anticoagulant is notrequired to be added into the container for measurement of cellfunctions, according to the first invention, is the case wheredetermination is made as to Regulated on Activation, Normal T Expressedand Secreted (RANTES) released by thrombin stimulating platelets.

Also, when needed, coagulant may be accommodated in the container formeasurement of cell functions according to the first invention. Such acase may arise when physiologically active substances are determinedthrough blood coagulation. Thrombin and the like may be cited asexamples of such coagulants.

An exemplary method of manufacturing the container for measurement ofcell functions, according to the first invention, will be now explainedreferring to the manufacture of the tubular container. It ismanufactured by adding anticoagulant or coagulant, depending upon theneed, into a tubular container which has a pressure reducible interiorand in which an amount of endotoxin, when extracted by collectingendotoxin-free water of a volume equal to the liquid volume to besubjected to measurement, is rendered at a level insufficient to induceproduction of physiologically active substances from blood cells;bringing the container into a predetermined vacuum condition; andplacing a stopper in the container.

As the above container, a closed-end tubular body, for example, ispreferred which is open at one end and closed at the opposite end. Anopening portion is preferably configured to be blocked by a stopperbody. Also, the container for measurement according to the firstinvention is preferably manufactured in as clean an atmosphere aspossible to avoid infection with endotoxin or various bacteria, andpreferably subjected to well-known sterilizing treatment aftercompletion of its manufacture, if possible.

In using the container for measurement of cell functions according tothe first invention, the above-specified container for measurement ofcell functions is first brought into communication with a blood vesselto allow blood to be suctioned into the above container for measurementof cell functions. In communicating the above blood vessel with theabove-specified container for measurement of cell functions, an injectorneedle may be employed which is utilized for conventional vacuum bloodcollecting techniques, i.e. a needle (generally called as a multipleinjector needle) which has on one side of a needle base a needle portionfor thrust into the blood vessel and on another side of the needle baseanother needle portion for thrust into the stopper in theabove-specified container, and in which the above-described needleportion for thrust into the blood vessel is communicated with anotherneedle portion for thrust into the stopper.

The blood obtained through use of the container for measurement of cellfunctions according to the first invention may be either transferred toanother container or subjected to measurement of physiologically activesubstances through successive use of the container. Also, the obtainedblood may be either transferred to another reactor or contacted withmaterial capable of inducing production of physiologically activesubstances, through successive use of the container as a reactor, formeasurement of produced physiologically active substances. In such acase where the container is employed as the reactor for contact with thematerial capable of inducing production of physiologically activesubstances, subsequent to blood collection in the container, such amaterial capable of inducing production of physiologically activesubstances is introduced into the container.

Determination of physiologically active substances in blood is effectedby determining the physiologically active substances in blood collectedthrough use of the container for measurement of cell functions. In thiscase, the blood collected through use of the container for measurementof cell functions according to the first invention is either transferredto another container or subjected to measurement of physiologicallyactive substances through successive use of the container. In such acase where determination of the physiologically active substances iscarried out through successive use of the container, the container,subsequent to blood collection, is generally left to stand orcentrifuged to separate hemocyte and plasmas containing physiologicallyactive substances which are quantitatively determined by reagents forrespective quantification thereof.

Cited as examples of the above physiologically active substances areTNF-α; interleukins such as IL-1β, IL-4, IL-5 and IL-6; interferons suchas INFα, INFβ and INFγ; colony-stimulating factors; chemotactic factorssuch as IL-8, RANTES and various cytokines; prostaglandins; PGE2 andPGI2; leukotrienes such as LTB4 and LTC4; various chemical mediatorssuch as; nitrogen monoxide, active oxygen, histamines, andplatelet-activating factor (PAF). Also cited are adhesion factors suchas soluble ICAM1, soluble cytokine receptors such as soluble IL-2receptor, matrix metallo proteinases, and intracellular granular enzymessuch as macrophage-specific elastase.

Cited as an exemplary method of determining the above-describedphysiologically active substances is an enzyme immunoassay whichutilizes monoclonal or polyclonal antibodies against their targetphysiologically active substances, peroxidases, enzymes such as alkalinephophatases, and chromophoric substrates of respective enzymes.

Cited as exemplary materials capable of inducing production ofphysiologically active substances are various microorganisms such asendotoxin, BCG dead bacterial and Corynebacteria; synthetic lipid A;pyran copolymer; lectins (such as phytohemagglutinin, concanavalin A,pokeweed mitogen); OK432 (Picibanil); PSK (Krestin); lentinan, zymosan;LPS (lipopolysaccharide); calcium ionophore; phorbol esters;immunoglobulin-fixed carriers; formylpeptides such asformyl-methionyl-leucyl-phenylalanine (FMLP); and various cytokines.

Also usable are specific antigens (for example, house dusts; miteantigens; various pollen antigens such as ragweed pollen extracts, cedarpollen extracts, rice extracts and the like; fungus antigens; parasiticantigens such as ascaris extracts and the like; food antigens such asovalbumin, wheat, soybean, lobster, crab, meat and the like; and wasptoxin) which are utilized for examination of allergens such as asthma,pollinosis, allergic rhinitis, atopic dermatitis, gastrointestinalallergy, parasitic allergy and the like.

Also cited are materials which fix the above-illustrated materialscapable of inducing production of physiologically active substances onvarious natural or synthetic high molecular weight materials, as bywell-known fixation techniques (such as covalent bonding, physicaladsorption and the like).

The shape of the above-described materials capable of inducingproduction of physiologically active substances is not particularlylimited, and may be in a liquid or particulate form, for example. Also,they may be fixed on carriers. In case of the liquid form, theabove-described materials capable of inducing production ofphysiologically active substances are generally diluted for use, such asby using, as a diluting liquid, a buffer solution such as phosphatebuffer, Hanks' buffer or the like, a normal culture medium such as MEM,RPMI-1640 or the like, a physiological saline (for example, manufacturedby Otsuka Seiyaku Co.), or water for injection (for example,manufactured by Otsuka Seiyaku Co.).

The existing form of the above-described material capable of inducingproduction of physiologically active substances may be either solid orliquid. In the event such a material capable of inducing production ofphysiologically active substances is water-soluble, it may be coated onan inner wall surface of the container or added to the container beforerendered into a powder form. For example, in the case where the materialcapable of inducing production of physiologically active substances isdiluted with water for injection, it is preferred to introduce thematerial capable of inducing production of physiologically activesubstances into the container for subsequent dry solidification thereof.In the case where the material capable of inducing production ofphysiologically active substances is water-insoluble, such awater-insoluble material may preferably be allowed to stay immersed inthe above-described diluting liquid, for example, since the possibleretention of bubbles on a surface of the material capable of inducingproduction of physiologically active substances is likely to causeexcessive hemolysis when it is brought into contact with blood, such asby tumble mixing, to result in adverse influence on a measurementsystem.

In the case where the material capable of inducing production ofphysiologically active substances is fixed to a carrier, if the fixingcarrier is a water-soluble material, such a water-soluble material maybe coated on an inner wall of the container or added to the containerbefore rendered into a powder form. If the fixing carrier is awater-insoluble material, such a water-insoluble material may preferablybe allowed to stay immersed in the above-described diluting liquid, forexample, since the possible retention of bubbles on a surface of thewater-insoluble material is likely to cause excessive hemolysis when itis brought into contact with blood, such as by tumble mixing, to resultin adverse influence on a measurement system.

It is preferred that the amount of the material capable of inducingproduction of physiologically active substances for addition to thecontainer is properly set to an optimum concentration level, dependingon the type of material capable of inducing production ofphysiologically active substances.

In the method for measurement of cell functions according to the firstinvention, in the case where another reactor is prepared separately fromthe container of the first invention, the shape of another reactor isnot particularly specified, and may be tubular as exemplified by bloodcollecting tubes, test tubes and the like, or plate-like as exemplifiedby microplates and the like. The reactor is preferably adapted such thatan amount of endotoxin, when extracted by collecting endotoxin-freewater of a volume equal to the liquid volume to be subjected tomeasurement, is rendered at a level insufficient to induce production ofphysiologically active substances from blood cells. Here, the meaning ofthe above-described liquid volume to be subjected to measurement isequivalent to that given above in explaining the container formeasurement of cell functions.

In the case where the reactor is prepared separately from the containerfor measurement of cell functions, as described above, such a reactorcan be employed to which the material capable of inducing production ofphysiologically active substances has been added, and which has beenbrought into a predetermined vacuum condition. For such a reactor, thecommunication thereof with the container for measurement of cellfunctions in which blood has been collected, such as provided by themultiple injector, facilitates transfer of the collected blood to thereactor. Also, the preloading of material capable of inducing productionof physiologically active substances simplifies a reaction process.

In the above case, it is preferred that the amount of material capableof inducing production of physiologically active substances for additionto the reactor is properly set to an optimum concentration level,depending on the type of material capable of inducing production ofphysiologically active substances. One exemplary method of manufacturingthis reactor is cited below. The material capable of inducing productionof physiologically active substances and, if necessary, anticoagulantare added to a tubular container which has a pressure reducibleinterior. Then, the reactor is brought into a predetermined vacuumcondition before the stopper is placed therein. The reactor for use inthe present invention is preferably manufactured in as clean anatmosphere as possible to avoid infection with endotoxin or variousbacteria, and is preferably subjected to well-known sterilizingtreatment after completion of its manufacture, if possible.

As the above-described reactor, a closed-end tubular body, for example,is preferred which is open at one end and closed at the opposite end.The opening portion is preferably configured to be successfully blockedby a stopper body. The above closed-end tubular body is preferably atest tube-like body suited for centrifugal operation, subsequent to thereaction, for measurement of the above-described material capable ofinducing production of physiologically active substances, and itspreferred size is 5-30 mm in outer diameter and 20-150 mm in height.

(Container for Measurement of Cell Functions according to The SecondInvention)

The container for measurement of cell functions according to the secondinvention is characterized in that a material, capable of inducingproduction of physiologically active substances in blood when itcontacts the blood, is accommodated therein in such a condition as beingcontactable with blood. This material capable of inducing production ofphysiologically active substances may also be hereinafter referred to asa physiologically active substance-inducing material. Cited as thephysiologically active substances can be those listed in the explanationof the first invention, and preferred are cytokines.

Also, a preferred physiologically active substance-inducing material isendotoxin. Endotoxin acts on monocytes and macrophages in blood topromote activation of these cells and induce production of cytokines.Cited as the above endotoxin is endotoxin consisting of cell-wallpolysaccharide (LPS) derived from microorganisms, for example. Also,materials can be employed which fixed endotoxin in various natural orsynthetic high molecular weight materials by a fixation technique.

The use amount of endotoxin is chosen such that a concentration ofendotoxin in a whole liquid (a sum of blood, an anticoagulant solution,an endotoxin dissolved solution and the others) when contacted withblood preferably falls within 0.6-100000 EU/ml, more preferably within0.8-80000 EU/ml. As the concentration falls below 0.6 EU/ml, the amountof induced TNFα, IL-1β and IL-6 possibly becomes excessively small. Asthe concentration goes beyond 100000 EU/ml, the amount of induced TNFα,IL-1β and IL-6 possibly becomes excessively small. This also adds to thecost.

In the container for measurement of cell functions according to thesecond invention, the above-described physiologically activesubstance-inducing material is rendered in such a condition as to becontactable with blood within the container. Such a condition as to becontactable with blood is, for example, where the physiologically activesubstance-inducing material is accommodated in the container.

The general shape of the physiologically active substance-inducingmaterial is either a powder form or a liquid form taken when theinducing material is dissolved in a solvent such as water. The conditionof the inducing material present in the container may be a solid, gel orliquid form. In the case of water-soluble inducing material, it may bedissolved in a suitable solvent for subsequent coating thereof on aninner wall surface of the container or addition to the container beforebrought into a powder form.

Any of buffers such as phosphate buffer, Hanks' buffer and the like, andnormal media such as MEM, RPM-1640 and the like can be utilized as theabove-described solvent, so far as it is a physiological buffer. Also, acommercially available water for injection (LPS-free water, manufacturedby Otsuka Seiyaku Co.) as well as a physiological saline (manufacturedby Otsuka Seiyaku Co.) can be utilized. Besides endotoxin, variousmaterials illustrated in the explanation of the first invention can alsobe utilized as the physiologically active substance-inducing materials.

In addition, the high molecular weight materials disclosed in a gazetteof Patent Laying-open No. Hei 6-209992 which have a surface roughnessmeasuring 0.2 μm-10 μm of a centerline average roughness Ra value and 5μm-200 μm of a mean spacing from peaks to valleys, the high molecularweight materials disclosed in a gazette of Patent Laying-open No. Hei7-67955 which have at least one chemical structure selected from thegroup consisting of a hydroxyl group, an amido skeleton, and an esterskeleton within a molecule, or high molecular weight materials having acationic functional group can be also used as the above-describedphysiologically active substance-inducing materials.

Also, among the above-described physiologically activesubstance-inducing materials, phytohemagglutinin is a preferredcytokine-inducing material. Phytohemagglutinin is tetrameric lectin, andits constitutive subunits include E subunit having hemagglutinativeactivity and L subunit having leukoagglutinative activity.Phytohemagglutinin-P (PHA-P) which is a tetramer consisting of E subunitand L subunit, and phytohemagglutinin-L (PHA-L) which is a tetramer of Lsubunit, are preferred as usable phytohemagglutinins. PHA-P and PHA-Lmay be used solely or in combination thereof.

Also, when comparison is made between PHA-P and PHA-L as to theirabilities to induce production of cytokines, with their use amountsbeing made equal, the induced amount exhibits about 10 times as high byPHA-L as by PHA-P. Accordingly, PHA-L is particularly preferred as thecytokine-inducing material. In case of PHA-L, a concentration ofphytohemagglutinin-L in a whole liquid (a sum of blood, an anticoagulantsolution, a PHA-L dissolved solution and the others), when brought intocontact with blood, is preferably rendered to fall within 0.1-100 μg/ml,and more preferably rendered to fall within 0.5-50 μg/ml. As the aboveconcentration falls below 0.1 μg/ml, the amount of induced TNFα andIL-1β possibly becomes excessively small, and as the concentration goesbeyond 50 μg/ml, the amount of induced TNFα and IL-1β possibly becomesexcessively small, which consequently adds to the cost.

Also, the above-described cytokine-inducing materials, other thanendotoxin, are preferably those containing substantially no endotoxin,so-called endotoxin-free materials.

Also, the amount of physiologically active substance-inducing materialincorporated in the container for use in the second invention must beregulated prior to use so as not to influence measured values of theabove inductively produced physiologically active substances. Asapparent from the below-described EXAMPLES, as the content of endotoxinsuch as LPS increases, a marked induction of cytokines is caused.Accordingly, in order to precisely perform measurement of cell functionsaccording to the second invention, the content of physiologically activesubstance-inducing material must be not greater than the levelinsufficient to induce cytokines.

The above-described endotoxin content, when endotoxin-free water of avolume equal to a liquid volume to be subjected to measurement iscollected in the container for subsequent extraction under agitation at37° C. for one hour, in the same manner as in the first invention, ispreferably rendered not greater than 0.5 EU (international endotoxinunit)/ml, as a concentration in the extracted solution.

Various techniques described in the explanation of the first inventioncan be employed as methods of removing or deactivating endotoxin.

Also, since the container for measurement of cell functions, accordingthe second invention, is employed for measuring functions of cellspresent in blood, anticoagulant may preferably be accommodated in theabove container to prevent blood coagulation.

The existing form, type and loading of the above anticoagulant are thesame as in the case of the container for measurement of cell functionsaccording to the first invention. Also, concerning the material types ofthe container for measurement of cell functions according to the presentinvention, those can be employed which are the same as of the containerfor measurement of cell function according to the first invention.

Furthermore, in the container for measurement of cell functionsaccording to the second invention, an interior thereof is preferablyvacuumed, thereby enabling ready suction of blood into the interior ofthe container for measurement of cell functions according to the secondinvention. In such a case, the use of a stopper is desired to maintainthe interior at a reduced pressure, as similar to the case of thecontainer for measurement of cell functions according to the firstinvention, and various materials illustrated in the explanation of thecontainer for measurement of cell functions according to the firstinvention can be exemplified as material types of the stopper.

The amount of blood collected in measuring cell functions using thecontainer for measurement of cell function according to the secondinvention is dependent on a volume of the container for measurement ofcell functions, but about 0.5-2 ml is sufficient when the employedcontainer for measurement of cell functions has a volume of 4-5 ml.

Cited as an exemplary method of manufacturing the container formeasurement of cell functions according to the second invention is amethod wherein the above-described physiologically activesubstance-inducing material, as well as anticoagulant, are added to acontainer which has a pressure reducible interior, the container isbrought into a predetermined vacuum condition, and the stopper is placedin the container.

As the above-described container, a closed-end tubular body, forexample, is preferred which is open at one end and closed at theopposite end. The opening portion is preferably configured to be blockedby a stopper body. More preferred as the above closed-end tubular bodyis the one suited for centrifugal operation performed subsequent to thereaction of inducing cytokines and for determining the amount of inducedcytokines, and its preferred size is 5-30 mm in outer diameter and about20-150 mm in height.

Also, the container for measurement of cell functions according to thefirst invention is preferably manufactured in as clean an atmosphere aspossible to avoid infection with endotoxin or various bacteria, and ispreferably subjected to well-known sterilizing treatment aftercompletion of its manufacture, if possible.

A method of measuring cell functions will now be explained utilizing thecontainer for measurement of cell functions in accordance with thesecond invention.

First, the above-described container for measurement of cell functionsis brought into communication with a blood vessel or a blood collectingcontainer so that a specimen blood is suctioned into the container formeasurement of cell functions. Moderate shaking is then applied to thecontainer for measurement of cell functions to contact the blood cellswith the above-described physiologically active substance-inducingmaterial for subsequent inductive reaction. As the reaction ceases, thecontainer is either left to stand or centrifuged to separate hemocyteand plasmas, and thereafter the cytokines in the plasmas arequantitatively determined by reagents capable of quantitating respectivecytokines.

The above-described technique to communicate the first container formeasurement of cell functions with the blood collecting container can beutilized to communicate the above-described blood collecting containerwith the above-described container for measurement of cell functions.

If the temperature at which the blood is reacted with the abovecytokine-inducing material becomes lower, the metabolic activity ofcells is possibly lowered to result in an excessively decreased amountof cytokines induced, and if it is elevated, the cell damage is possiblycaused to result in an excessively decreased amount of cytokinesinduced. Accordingly, it is controlled preferably at 26-45° C., morepreferably at 30-42° C.

If the time period during which the blood is reacted with the abovecytokine-inducing material is shortened, the amount of cytokines inducedpossibly becomes excessively small, and if it is excessively prolonged,the production of measurement results is delayed. Also, the amount ofcytokines induced shows a trend of gradually decreasing from a peakwhich takes place in about 4 hours. The preferred time period is thus1-6 hours, more preferably 2-4 hours.

In the method of measuring cell functions utilizing the container formeasurement of cell functions according to the second invention, it ismost preferred that a whole blood collected in the container formeasurement of cell functions is cultured at 30-40° C. for 2-6 hours toinduce cytokines.

The enzyme immunoassay described in the explanation of the container formeasurement of cell functions according to the first invention can beutilized to quantitatively determine induced cytokines.

One embodiment of a technique to measure cell functions using thecontainer for measurement of cell functions according to the secondinvention will be now explained in detail. First, the above-describedcytokine-inducing material is reacted with blood in the above-describedcontainer for measurement of cell functions to induce cytokines. As theinduction completes, the container for measurement of cell functions iscentrifuged at 1200 G to separate hemocyte components and plasmacomponents. Next, the separated plasmas are added using a pipette into awell of a microplate on which monoclonal anti-cytokine antibodies havebeen fixed for subsequent reaction at 37° C. for 2 hours. Then, theplasma solution after reaction was removed by means of suction removalor the like, and in addition, the well is washed with a neutral pHcleaning buffer containing a nonionic surfactant, such as Tween 20, tofurther remove unreacted components. Horseradish peroxidases-fixedpolyclonal anti-cytokine antibodies are then pipette added to the wellfor reaction at 37° C. for 1 hour. The well is then washed with theabove cleaning buffer to remove unreacted horseradish peroxidases, andthereafter a substrate solution containing hydrogen peroxide andtetramethylbenzidine is added for reaction for 5˜10 minutes. An 1 Msolution of sulfuric acid is added to discontinue the reaction beforedetermining a color produced in the substrate due to an enzyme reactionfrom absorbance at 450 nm. The determined value is evaluated against acalibration curve prepared by using cytokines of known concentration todetermine the level of cytokines induced.

(Kit for Measurement of Cell Functions)

Each of the containers for measurement of cell functions according tothe first and second inventions can be combined with a reagent capableof quantitating physiologically active substances, such as an enzymeimmunoassay reagent, to provide a usable kit for measurement of cellfunctions. That is, a kit for measurement of cell functions can beprovided which has the container for measurement of cell functionsaccording to the first invention and the reagent capable ofquantitatively determining induced physiologically active substances. Inaddition, a kit for measurement of cell functions can also be providedwhich has the container for measurement of cell functions according tothe second invention and the reagent capable of quantitativelydetermining physiologically active substances induced.

The technique referred to above as one embodiment of measuring cellfunctions with the use of the container for measurement of cellfunctions according to the second invention can be similarly employedwhen using the above-described kits for measurement of cell functions,for example.

(Preferred Anticoagulants)

In the present invention, the amount of material capable of inducingproduction of physiologically active substances contained in theabove-described anticoagulant is desirably controlled at a levelinsufficient to produce the physiologically active substances from bloodcells when mixed with blood. That is, the reduction in amount of thephysiologically active substances originally contained in theanticoagulant effectively restrains the occurrence of unnecessarystimulation given to the collected blood prior to assaying. Theproduction of physiologically active substances in the collected blooddue to the action of the anticoagulant is thus regulated, so that thedetermination of various physiologically active substances, measurementof cell functions, and determination of surface antigens of blood cellscan be carried out more precisely. Also, a specimen of blood can bepreserved for a prolonged period from collection till assaying.

Again in such a case, the physiologically active substance-inducingmaterials may be those described above, and preferred one is endotoxin.The increased content of endotoxin causes production of the above-listedcytokines, such as TNFα, IL-1β, IL-6 and the others, to interfere with aprecise measurement. Accordingly, the endotoxin content in theanticoagulant is desirably regulated to a level insufficient to producecytokines, as physiologically active substances, in collected blood.

As will become apparent from Examples described hereinafter, theproduction of cytokines, such as TNFα, IL-1β, IL-6 and the others, ispossibly induced if the endotoxin content in anticoagulant goes beyond0.5 EU/ml in reactive blood. It is accordingly desired to regulate theendotoxin content in anticoagulant so that the endotoxin content in areactive liquid does not exceed 0.5 EU/ml.

The amount of the above-described anticoagulant is dependent upon theblood amount to be collected, but is generally 0.5-5 mg/ml in blood, ifsodium ethylenediamine tetraacetate is used, 3-5 weight % in blood, ifsodium citrate is used, and 4-50 U/ml in blood, if heparin sodium isused.

Accordingly, it is preferred in the present invention that the endotoxincontent in anticoagulant may be suitably selected depending on theamounts respectively of anticoagulant and collected blood, such that theendotoxin content in blood collected results in a level not exceeding0.5 EU/ml.

For example, when 1 ml of blood is collected for examination, heparinsodium, if selected for use, is added generally in an amount of 4-50U/ml, and accordingly the preferred endotoxin content thereof is notgreater than 00.125 EU/heparin unit, more preferably not greater than0.01 EU/heparin unit.

The variety of techniques described in the explanation of the firstinvention for removing or deactivating endotoxin, for example, can beemployed to manufacture anticoagulant containing a reduced amount ofendotoxin.

However, the inactivation of endotoxin with heat, acid or alkalitreatment sometimes accompanies deactivation of a certain anticoagulantitself. Accordingly, the ultrafiltration using a membrane or the removalusing an adsorbent is preferred.

(Measurement of Cell Functions)

The term “measurement of cell functions” as used in the presentinvention is intended to include a method of directly measuringfunctions of a blood cell which may be classified into an erythrocyte, aplatelet and a leukocyte, a method of evaluating cell functions throughdetermination of physiologically active substances, and measurement ofblood cell surface antigens.

The measurement of functions of the blood cell which is classified intothe above-mentioned erythrocyte, platelet and leukocyte includehemagglutination, platelet agglutination, leukocyte migration, leukocytemigration inhibition test, leukocyte nitroblue tetrazolium reduction,leukocyte phagocytic activity, lymphocyte transformation, lymphocytecytotoxic test, antibody-dependent cell-mediated cytotoxic activitytest, cytokines-producing capacity, histamine release test and the like,for example.

Also, the measurement of cell surface antigens refers to measurement ofcell surface antigens through a rosette formation test or flowcytometry, and includes measurement of Fc receptors and various CDantigens, for example.

(Kit for Measurement of Cell Functions according to The Third Invention)

The kit for measurement of cell functions according to the thirdinvention has the above-described container for measurement of cellfunctions according to the first invention, the container formeasurement of cell functions according to the second invention and thereagent.

The details of usable containers for measurement of cell functionsaccording to the first and second inventions are hereinbefore described.

In order to measure the cell functions using the kit for measurement ofcell functions according to the third invention, the container formeasurement of cell functions according to the second invention iscommunicated with a blood collecting container, a specimen of blood isintroduced into the container for measurement of cell functionsaccording to the second invention, and then the container formeasurement of cell functions according to the second invention isshaken to react the blood cells with the physiologically activesubstance-inducing material.

Also, in order to obtain a control value, the blood collecting containeris communicated with the container for measurement of cell functionsaccording to the first invention to introduce the specimen of blood intothe container for measurement of cell functions according to the firstinvention.

Next, the containers for measurement of cell functions according to thefirst and second inventions, into which blood has been introduced insuch a manner as described above, are either left to stand orcentrifuged to separate hemocyte and plasmas, and the physiologicallyactive substances in plasmas in respective containers for measurement ofcell functions according to the first and second inventions areseparately quantitatively determined by the use of the first and secondenzyme immunoassay reagents having respective measurement sensitivitiesdifferent from each other.

The communication of the blood collecting container with the respectivecontainers for measurement of cell functions according to the first andsecond inventions can be achieved using the above-described technique.

As the reaction temperature of the blood and endotoxin in the containerfor measurement of cell functions according to the second inventiondecreases, reduced metabolic activity of cells, as well as reducedinduction of cytokines, result. As it increases, cell injury and reducedinduction of cytokines result. Accordingly, it is preferably 26-45° C.,more preferably 30-42° C.

In view of the efficient production of cytokines and prevention ofexcessive hemolysis, the reaction period of the blood and endotoxin ispreferably 1-12 hours, more preferably 2-6 hours.

In the kit for measurement of cell functions according to the presentinvention, the physiologically active substances in blood collected inthe container for measurement of cell functions according to the secondinvention, that is, the physiologically active substances produced inblood due to its reaction with the physiologically activesubstance-inducing material is quantitatively determined by the secondenzyme immunoassay reagent. The physiologically active substancesproduced in blood due not to its reaction with the physiologicallyactive substance-inducing material is quantitatively determined by thefirst enzyme immunoassay reagent. The resulting differential allowsdetermination of the exact production of physiologically activesubstances induced by the physiologically active substance-inducingmaterial.

Generally, the amount of cytokines in blood (the amount of cytokines inblood collected in the first container for measurement of cellfunctions) prior to its reaction with endotoxin is several pg/ml—severalhundreds pg/ml, and the amount of cytokines in blood after its reactionwith endotoxin ranges from several hundreds pg/ml to several thousandspg/ml or higher.

However, there exists no such a reagent having a measurement sensitivitysufficient to quantitate cytokines over a wide range of severalpg/ml—several thousands pg/ml. Accordingly, for determination ofcytokines in an amount of 1,000 pg/ml or higher, plasmas have beendiluted with a suitable diluting solution. However, the dilutingoperation of plasmas is complicated, separately requires the dilutingsolution and a dilution container, and accordingly leads to a markedlyprolonged measurement period. Also, the measured value is obtained bymultiplying the degrees of dilution, which has brought about a problemof lowering accuracy of the measured value.

In contrast, in accordance with the third invention, the amount ofcytokines in blood (i.e., the amount of cytokines in blood collected inthe first container for measurement of cell functions) prior to itsreaction with endotoxin can be determined by the first enzymeimmunoassay reagent of high sensitivity, such as of a measurementsensitivity of 10-1,000 pg/ml, while the amount of cytokines in blood(i.e., the amount of cytokines in blood collected in the secondcontainer for measurement of cell functions) after its reaction withendotoxin can be determined by the second enzyme immunoassay reagent oflow sensitivity, such as of a measurement sensitivity of 500- about10,000 pg/ml. The above-described, complicated diluting operation canthus be eliminated.

Also, if the above-described cytokine consists of TNFα or IL-β, thepreferred sensitivity of the first enzyme immunoassay reagent is 10-500pg/ml and that of the second enzyme immunoassay reagent is 500-10,000pg/ml, if LI-6, the preferred sensitivity of the first enzymeimmunoassay reagent is 10-1,000 pg/ml and that of the second enzymeimmunoassay reagent is 1,000-20,000 pg/ml.

Each of the above-described first and second enzyme immunoassay reagentsis comprised of either monoclonal or polyclonal antibody against itstarget cytokines, enzymes such as peroxidases or alkaline phosphatases,and chromophoric substrates in respective enzymes. The sandwich enzymeimmunoassay, wherein the monoclonal antibody against its targetcytokines is prefixed on a solid surface as of a microplate, ispreferred since it does not require the fixation prior to measurementand is excellent in reproducibility.

A technique of fixing the monoclonal antibodies on the solid surfacesmay be arbitrarily chosen from the known physical adsorption or chemicalbonding technique, but the physical adsorption is preferred for itssimplified operation.

The preparation of the first and second enzyme immunoassay reagentshaving different sensitivities can be accomplished by selectivelyadjusting concentrations respectively of monoclonal or polyclonalantibodies against their target cytokines, enzymes such as peroxidasesor alkaline phosphatases, and chromophoric substrates in respectiveenzymes.

For example, in the case where the sandwich enzyme immunoassay isemployed, the amount of the above-mentioned monoclonal antibody againstits target cytokines for fixation on the microplate may be adjusted, sothat the first and second enzyme immunoassay re agents can be preparedwhich have different sensitivities. That is, the amount of targetcytokines bindable to the monoclonal antibody changes depending on theamount of monoclonal antibody fixed on the microplate surface, whichenables preparation of reagents having different measurementsensitivities.

Such different measurement sensitivities can also be accomplished bypreparing different concentrations of either enzyme-labelled monoclonalantibody or enzyme-labelled polyclonal antibody against its targetcytokines, which differ from the target cytokines of the fixedmonoclonal antibody, as a reagent for detecting cytokines bound to thefixed monoclonal antibody.

There also is a method in which a specific binding mode such asavidin-biotin is incorporated in the above-described enzyme immunoassaysystem, e.g. a method in which biotin-labelled antibody or/andavidin-labelled enzyme is used as an alternative to enzyme-labelledantibody against its target cytokines which differ from the targetcytokines of the fixed monoclonal antibody.

An exemplary preparation of the monoclonal antibody-fixed microplateswill be now explained which can be employed for the present first andsecond enzyme immunoassay reagents having different measurementsensitivities.

First, the specific monoclonal antibody against its target cytokines issuch dissolved in a buffer solution, such as phosphate buffer, as toprepare two types of dilute solutions of concentration levels differentfrom each other (each concentration level prepared is suitably selecteddepending upon the magnitude of binding constant of the employedmonoclonal antibody and its target cytokines).

Each of the two types of monoclonal antibody dissolved solutions isadded in a given amount to a microplate for incubation at 2-8° C. for aday and a night. Subsequent washing with a neutral pH cleaning buffercontaining a nonionic surfactant such as Tween 20, a given amount of 1-4weight % bovine serum albumin dissolved phosphate buffer solution isadded to the microplate for incubation at 37° C. for 2 hours. Afterremoval of liquids from the microplate, it is dried at room temperature.

One embodiment of measuring cell function using the kit for measurementof cell functions according to the third invention will be now explainedin detail.

First, blood is introduced into each of the first and second containersfor measurement of cell functions for incubation at 37° C. for 4 hours.Each container is centrifuged at 1600 G to allow hemocyte and plasmas toseparate. Then, the separated plasmas are added to wells of respectivemicroplates to which the monoclonal antibody against its targetcytokines has been fixed in two different concentration levels, whichhave been blocked at unadsorbed cites by the bovine serum albumins, andwhich have been dried, for reaction at 37° C. for 2 hours. Next, theplasma solution after reaction is discarded by means of suction removaland the like, followed by washing the wells with the neutral pH cleaningbuffer containing a nonionic surfactant such as Tween 20 to furtherremove unreacted components. Thereafter, the horseradishperoxidase-fixed polyclonal antibody against the above-specifiedcytokines is added to the wells for reaction at. 37° C. for 1 hour. Inorder to remove the unreacted portion of horseradish peroxidase-fixedpolyclonal antibody, the wells are washed with the aforementionedcleaning buffer, and thereafter a substrate solution containing hydrogenperoxide and tetramethyl-benzidine is added to the wells for reactionfor 5-10 minutes. A 2 M solution of sulfuric acid is then added, thereaction is terminated, and a color produced in the substrate as aresult of the enzyme reaction is measured from absorbance at 450 nm. Thecomparison of the measured value with a calibration curve preparedagainst the above-specified cytokines of a known concentrationquantitatively determines the cytokines in blood as treated in each ofthe first and second containers for measurement of cell functions.

(Method for Measurement of Cell Functions according to The FourthInvention)

The method for measurement of cell functions according to the fourthinvention is characterized by introducing blood into the first or secondcontainer for measurement of cell functions and measuring the cellfunctions. In this case, the container for measurement of cell functionspreferably accommodates, in advance, the above-specified anticoagulantwhich contains endotoxin in such a limited concentration as not toinduce blood cells to produce physiologically active substances whenmixed with blood.

One embodiment of the method for measurement of cell functions accordingto the fourth invention is explained below.

The anticoagulant, e.g. heparin sodium is accommodated in an injectorhaving a blood-collecting needle in a typical amount of 10 U/ml perblood to be collected, before blood is collected from an examined personinto the injector. Alternatively, the anticoagulant may be accommodatedin a vacuum blood-collecting tube in the same amount as above beforeeffecting collection of blood. Next, this blood is centrifuged at 1600G, and the amount of TNFα, as illustrative of the physiologically activesubstance produced from blood cells, in plasma is determined using theenzyme immunoassay.

Also, in a more particular aspect of the method for measurement of cellfunctions according to the fourth invention, the container formeasurement of cell functions according to the second invention isutilized. In such a case, the physiologically active substance-inducingmaterial is arranged within the container so as to be contactable withblood. Accordingly, if brought into contact with blood, thephysiologically active substance-inducing material reacts with blood toinduce production of the physiological active substances, asabove-described in the explanation of the container for measurement ofcell functions according to the second invention. The physiologicalactive substances induced can be determined using the aforementionedtechniques.

A preferred temperature at which the physiologically active substancesare induced is in the range of 26-45° C., and a preferred time period ofinducing the physiologically active substances is 1-6 hours.

Also, the physiologically active substances induced can bequantitatively determined by reagents capable of quantitating thereof,e.g. enzyme immunoassay reagents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing measurement results obtained in Examples 1-4and Comparative Examples 2-4, wherein the abscissa indicates theconcentration of LPS and the ordinate indicates the amount of TNFαinduced.

FIG. 2 is a graph showing measurement results obtained in Examples 1-4and Comparative Examples 2-4, wherein the abscissa indicates theconcentration of LPS and the ordinate indicates the amount of IL-1βinduced.

FIG. 3 is a graph showing measurement results obtained in Examples 1-4and Comparative Examples 2-4, wherein the abscissa indicates theconcentration of LPS and the ordinate indicates the amount of IL-6induced.

FIG. 4 is a graph showing measurement results obtained in Examples 5-10,wherein the abscissa indicates the concentration of LPS and the ordinateindicates the amount of TNFα or IL-1β induced.

FIG. 5 is a graph showing measurement results obtained in Examples11-16, wherein the abscissa indicates the reaction temperature and theordinate indicates the amount of TNFα or IL-1β induced.

FIG. 6 is a graph showing measurement results obtained in Examples17-21, wherein the abscissa indicates the reaction time and the ordinateindicates the amount of TNFα or IL-1β induced.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will be now explained in detail by citing Examplesof the present invention and Comparative Examples. It should beunderstood that the present invention is not limited to the Exampleswhich follow.

Examples 1-4, Comparative Examples 1-4

(1) Method for Manufacturing A Container for Measurement of CellFunctions:

A 4 ml blood-collecting tube (12.6×75 mm in diameter) made ofpolyethylene terephthalate was well washed 10 times with 4 ml ofendotoxin-free water (manufactured by Otsuka Seiyaku Co.). 0.05 ml of aphysiological saline for injection (manufactured by Otsuka Seiyaku Co.)containing heparin sodium (manufactured by Novo.Nordisk A/S Co., productname: Novo.Heparin # 1000) in concentration of 200 U/ml was added to theblood-collecting tube.

E. coli UKT-B derived endotoxin (standard product Lot. 8920 of Japanesepharmacopeia) was dissolved in a physiological saline for injection(manufactured by Otsuka Seiyaku Co.) for subsequent stepwise dilution.As each dilution step completed, 0.05 ml of the resulting endotoxindissolved physiological sline is added to every two of the heparinincorporated blood-collecting tube, so that the blood-collecting tubeswere prepared which respectively contained endotoxin in concentrationsof 0 EU/ml (Comparative Example 1), 1 EU/ml (Comparative Example 2), 2EU/ml (Comparative Example 3), 5 EU/ml (Comparative Example 4), 10 EU/ml(Example 1), 20 EU/ml (Example 2), 50 EU/ml (Example 3) and 100 EU/ml(Example ) per a whole solution summing the heparin dissolvedphysiological saline and the endotoxin dissolved physiological saline.

Next, a butyl rubber-made stopper, which was configured to fit into thetube and had been previously washed with an endotoxin-free water(manufactured by Otsuka Seiyaku Co.), was lightly placed at an openingof each blood-collecting tube so as not to tightly close the opening.Then, every blood-collecting tube was placed within a vacuum container.When an interior of the container was gradually reduced to a pressure of570 mmHg, the opening of each blood-collecting tube was tightly closedby the stopper. The blood-collecting tubes thus prepared were employedas the containers for measurement of cell functions in Examples 1-4 andComparative Examples 1-4, respectively.

(2) Determination of Endotoxin Contents in The Blood-Collecting Tubes(Containers):

The endotoxin-free water (manufactured by Otsuka Seiyaku Co.) wasintroduced into an injector with a needle. The needle of injector wasthrust into the butyl rubber-made stopper, as placed on each of the fourvacuumed blood-collecting tubes which were obtained in the above (1) ascontaining heparin solely, to inject 0.9 ml of the endotoxin-free water(manufactured by Otsuka Seiyaku Co.) into each of the blood-collectingtubes, followed by agitation at 37° C. for 1 hour to extract endotoxin.Then, the endotoxin content in the extracted liquid was determinedthrough a synthesized chromophoric substrate technique using a kit forthe determination of endotoxin, ENDOSPECIE ES6 (product name)manufactured by Seikagaku Kogyo Co.

The results indicated the endotoxin content in the extracted liquid asbeing not greater than 0.05 EU/ml for each of the tested fourblood-collecting tubes.

(3) Method for Determining TNFα, IL-1β and IL-6 Inducing Activities:

Using an injector with a needle, heparinized blood was collected from ausually healthy volunteer. The needle of injector was thrust into thebutyl rubber-made stopper, as placed on each of the vacuumedblood-collecting tubes which were obtained in the above (1) ascontaining endotoxin in various concentrations, to inject 0.9 ml of thecollected specimen blood into each of the blood-collecting tubes. Next,each of the blood-collecting tubes was mounted to a rocker platform fortumble mixing in a thermostatic chamber preheated to a temperature of37° C. for subsequent tumble mixing for 4 hours. As the intimate mixingwas completed, each blood-collecting tube was centrifuged at 1600 G at4° C. for 10 minutes to collect a supernatant plasma. The collectedplasma was determined for contents of cytokines (pg/ml), i.e. respectivecontents of TNFα, IL-1β and IL-6, using an enzyme immunoassay kit whichutilized respective monoclonal antibodies against them.

PREDICTA Human TNF-α ELISA KIT (limit of detection: 35 pg/ml), PREDICTAHuman IL-1β ELISA KIT (limit of detection: 15 pg/ml) and PREDICTA HumanIL-6 ELISA KIT (limit of detection: 35 pg/ml) were employed (allmanufactured by Genzyme Inc.) to determine the productions in weight ofTNFα, IL-1β and IL-6, respectively. The determination was effected usingn=3 for each. The results are shown in FIGS. 1-3.

In all Figures, the unit EU/ml used for the LPS concentration on theabscissa indicates the endotoxin concentration in a whole solution whenin contact with blood, and the induced amount (pg/ml) on the ordinateindicates an average value of the values obtained from the twoblood-collecting tubes for each cytokine concentration in plasma. Asapparent from the results, endotoxin at a concentration of 0.6 EU/ml orhigher clearly induces productions of TNFα, IL-1β and IL-6. The resultsfrom the case where the endotoxin concentration was 0 EU/ml (ComparativeExample 1) are not shown in FIGS. 1-3, since in this case, theconcentration of each cytokine produced was below the limit of detectionin the determination thereof.

Examples 5-10

(1) Method for Manufacturing A Container for Measurement of CellFunctions:

A 4 ml blood-collecting tube (12.6×75 mm in diameter) made ofpolyethylene terephthalate was well washed 10 times with 4 ml ofendotoxin-free water (manufactured by Otsuka Seiyaku Co.). 0.05 ml of aphysiological saline for injection (manufactured by Otsuka Seiyaku Co.)containing heparin sodium (manufactured by Novo.Nordisk A/S Co., productname: Novo.Heparin # 1000) in a concentration of 200 U/ml was added tothe tube.

E. coli 055:B5 derived endotoxin (manufactured by LBL corp.) wasdissolved in a physiological saline for injection (manufactured byOtsuka Seiyaku Co.) for subsequent stepwise dilution. As each dilutionstep completed, 0.05 ml of the resulting endotoxin dissolvedphysiological sline is added to every two of the heparin incorporatedblood-collecting tube, so that the blood-collecting tubes were preparedwhich respectively contained endotoxin in concentrations of 8 EU/ml(Example 5), 80 EU/ml (Example 6), 800 EU/ml (Example 7), 8000 EU/ml(Example 8), 80000 EU/ml (Example 9) and 800000 EU/ml (Example 10) per awhole solution summing the heparin dissolved physiological saline andthe endotoxin dissolved physiological saline.

Next, a butyl rubber-made stopper, which was configured to fit into thetube and had been previously washed well with an endotoxin-free water(manufactured by Otsuka Seiyaku Co.), was lightly placed at an openingof each blood-collecting tube so as not to tightly close the opening.Then, every blood-collecting tube was placed within a vacuum container.When an interior of the container was gradually reduced to a pressure of570 mmHg, the opening of each blood-collecting tube was tightly closedby the stopper. The blood-collecting tubes thus prepared were employedas the containers for measurement of cell functions in Examples 5-10,respectively.

(2) Determination of Endotoxin Contents in The Blood-Collecting Tubes(Containers):

The endotoxin-free water (manufactured by Otsuka Seiyaku Co.) wasintroduced into an injector with a needle. The needle of injector wasthrust into the butyl rubber-made stopper, as placed on each of the fourvacuumed blood-collecting tubes which were obtained in the above (1) ascontaining heparin solely, to inject 0.9 ml of the endotoxin-free water(manufactured by Otsuka Seiyaku Co.) into each of the blood-collectingtubes, followed by agitation at 37° C. for 1 hour to extract endotoxin.Then, the endotoxin content in the extracted liquid was determinedthrough a synthesized chromophoric substrate technique using a kit forthe determination of endotoxin, ENDOSPECIE ES6 (product name)manufactured by Seikagaku Kogyo Co.

The results indicated the endotoxin content in the extracted liquid asbeing not greater than 0.05 EU/ml for each of the tested fourblood-collecting tubes.

(3) Method for Determining TNFα and IL-1β Inducing Activities:

Using an injector with a needle, heparinized blood was collected from ausually healthy volunteer. The needle of injector was thrust into thebutyl rubber-made stopper, as placed on each of the vacuumedblood-collecting tubes which were obtained in the above (1) ascontaining endotoxin in various concentrations, to inject 0.9 ml of thecollected specimen blood into each of the blood-collecting tubes. Next,each of the blood-collecting tubes was mounted to a rocker platform fortumble mixing in a thermostatic chamber preheated to a temperature of37° C. for subsequent tumble mixing for 4 hours. As the intimate mixingwas completed, each blood-collecting tube was centrifuged at 1600 G at4° C. for 10 minutes to collect a supernatant plasma. The collectedplasma was determined for contents (pg/ml) of TNFα and IL-1β in the samemanner as employed in Example 1.

The results are shown in FIG. 4. In FIG. 4, the unit EU/ml used for theLPS concentration on the abscissa indicates the endotoxin concentrationin blood (in a whole solution) when in contact with blood, and theinduced amount (pg/ml) on the ordinate indicates an average value of thevalues obtained from the two blood-collecting tubes for each of the TNFαand IL-1β concentrations in plasma. As apparent from the results, theendotoxin concentration in the range of 0.8-80000 EU/ml inducesproductions of TNFα and IL-1β. Also, when the endotoxin concentration isin the range of of 8-800 EU/ml, the amounts of TNFα and IL-1β inducedare indicated as both increasing sluggishly. When the endotoxinconcentration reaches 8000 EU/ml, the amounts of TNFα and IL-1β inducedboth indicated a further increase. However, when the endotoxinconcentration reaches 80000 EU/ml, the amounts of TNFα and IL-1β inducedeach becomes smaller than when the endotoxin concentration is 8000EU/ml.

The use of endotoxin, as illustrative of the cytokine-inducing material,in the range of 10-200 μg/ml (about 80000-2000000 EU/ml as converted tothe endotoxin concentration) has been already described in the gazetteof Patent Laying-open No. Hei 1-503331. However, the present inventionenables use of endotoxin in lower concentrations, so that the cytokineinduction via plural mechanisms does not occur which is believed due tohigher concentrations of endotoxin. Therefore, in accordance with thepresent invention, it becomes possible to induce cytokines whichaccurately reflects the patients' morbidities.

Examples 11-16

(1) Method for Manufacturing A Container for Measurement of CellFunctions:

Each of 4 ml blood-collecting tubes (12.6×75 mm in diameter) made ofpolyethylene terephthalate was well washed 10 times with 4 ml ofendotoxin-free water (manufactured by Otsuka Seiyaku Co.). 0.05 ml of aphysiological saline for injection (manufactured by Otsuka Seiyaku Co.)containing heparin sodium (manufactured by Novo.Nordisk A/S Co., productname: Novo-Heparin # 1000) in a concentration of 200 U/ml was added toeach tube.

E. coli 055:B5 derived endotoxin (manufactured by LBL corp.) wasdissolved in a physiological saline for injection (manufactured byOtsuka Seiyaku Co.) to a concentration of 1600 EU/ml. 0.05 ml of theresulting endotoxin dissolved physiological sline solution was added toeach of the above-prepared, heparin incorporated blood-collecting tubes.

Next, a butyl rubber-made stopper, which was configured to fit into thetube and had been previously washed well with an endotoxin-free water(manufactured by Otsuka Seiyaku Co.), was lightly placed at an openingof each blood-collecting tube so as not to tightly close the opening.Then, every blood-collecting tube was placed within a vacuum container.When an interior of the container was gradually reduced to a pressure of570 mmHg, the opening of each blood-collecting tube was tightly closedby the stopper. The blood-collecting tubes thus prepared were employedas the containers for measurement of cell functions in Examples 11-16,respectively.

(2) Determination of Endotoxin Contents in The Blood-Collecting Tubes(Containers):

The endotoxin-free water (manufactured by Otsuka Seiyaku Co.) wasintroduced into an injector with a needle. The needle of injector wasthrust into the butyl rubber-made stopper, as placed on each of the fourvacuumed blood-collecting tubes which were obtained in the above (1) ascontaining heparin solely, to inject 0.9 ml of the endotoxin-free water(manufactured by Otsuka Seiyaku Co.) into each of the blood-collectingtubes, followed by agitation at 37° C. for 1 hour to extract endotoxin.Then, the endotoxin content in the extracted liquid was determinedthrough a synthesized chromophoric substrate technique using a kit forthe determination of endotoxin, ENDOSPECIE ES6 (product name)manufactured by Seikagaku Kogyo Co.

The results indicated the endotoxin content in the extracted liquid asbeing not greater than 0.05 EU/ml for each of the tested fourblood-collecting tubes.

(3) Method for Determining TNFα and IL-1β Inducing Activities:

Using an injector with a needle, heparinized blood was collected from ausually healthy volunteer. The needle of injector was thrust into thebutyl rubber-made stopper, as placed on each of the vacuumedblood-collecting tubes which were obtained in the above (1), to inject0.9 ml of the collected specimen blood into each of the blood-collectingtubes. Next, two of the blood-collecting tube in which the specimenblood had been collected were mounted to each of the rocker platformsfor tumble mixing respectively preheated to a temperature of 25° C.(Example 11), 30° C. (Example 12), 33° C. (Example 13), 37° C. (Example14), 40° C. (Example 15), and 45° C. (Example 16) in a thermostaticchamber for subsequent tumble mixing for 4 hours. As the intimate mixingwas completed, each blood-collecting tube was centrifuged at 1600 G at4° C. for 10 minutes to collect a supernatant plasma. The collectedplasma was determined for contents (pg/ml) of TNFα and IL-1β in the samemanner as employed in Example 1.

The results are shown in FIG. 5. In FIG. 5, the reaction temperature onthe abscissa indicates the temperature at which the thermostatic chamberwas set, and the induced amount (pg/ml) on the ordinate indicates anaverage value of the values obtained from the two blood-collecting tubesfor each of the TNFα and IL-1β concentrations in plasma.

Examples 17-21

(1) Method for Manufacturing A Container for Measurement of CellFunctions:

Each of 4 ml blood-collecting tubes (12.6×75 mm in diameter) made ofpolyethylene terephthalate was well washed 10 times with 4 ml ofendotoxin-free water (manufactured by Otsuka Seiyaku Co.). 0.05 ml of aphysiological saline for injection (manufactured by Otsuka Seiyaku Co.)containing heparin sodium (manufactured by Novo.Nordisk A/S Co., productname: Novo-Heparin # 1000) in a concentration of 200 U/ml was added toeach tube.

E. coli 055:B5 derived endotoxin (manufactured by LBL corp.) wasdissolved in a physiological saline for injection (manufactured byOtsuka Seiyaku Co.) to a concentration of 1600 EU/ml. 0.05 ml of theresulting endotoxin dissolved physiological sline solution was added toeach of the above-prepared, heparin incorporated blood-collecting tubes.

Next, a butyl rubber-made stopper, which was configured to fit into thetube and had been previously washed well with an endotoxin-free water(manufactured by Otsuka Seiyaku Co.), was lightly placed at an openingof each blood-collecting tube so as not to tightly close the opening.Then, every blood-collecting tube was placed within a vacuum container.When the interior of the container was gradually reduced to a pressureof 570 mmHg, the opening of each blood-collecting tube was tightlyclosed by the stopper. The blood-collecting tubes thus prepared wereemployed as the containers for measurement of cell functions in Examples17-21, respectively.

(2) Determination of Endotoxin Contents in The Blood-Collecting Tubes(Containers):

The endotoxin-free water (manufactured by Otsuka Seiyaku Co.) wasintroduced into an injector with a needle. The needle of injector wasthrust into the butyl rubber-made stopper, as placed on each of the fourvacuumed blood-collecting tubes which were obtained in the above (1) ascontaining heparin solely, to inject 0.9 ml of the endotoxin-free water(manufactured by Otsuka Seiyaku Co.) into each of the blood-collectingtubes, followed by agitation at 37° C. for 1 hour to extract endotoxin.Then, the endotoxin content in the extracted liquid was determinedthrough a synthesized chromophoric substrate technique using a kit forthe determination of endotoxin, ENDOSPECIE ES6 (product name)manufactured by Seikagaku Kogyo Co.

The results indicated the endotoxin content in the extracted liquid asbeing not greater than 0.05 EU/ml for each of the tested fourblood-collecting tubes.

(3) Method for Determining TNFα and IL-1β Inducing Activities:

Using an injector with a needle, haparinized blood was collected from ausually healthy volunteer. The needle of injector was thrust into thebutyl rubber-made stopper, as placed on each of the 12 vacuumedblood-collecting tubes which were obtained in the above (1), to inject0.9 ml of the collected specimen blood into each of the blood-collectingtubes. Next, the blood-collecting tubes in which the specimen blood hadbeen collected were mounted to a rocker platform for tumble mixingpreheated to a temperature of 37° C. in a thermostatic chamber forsubsequent tumble mixing for 30 minutes (Example 17), 2 hours (Example18), 4 hours (Example 19), 6 hours (Example 20), and 24 hours (Example21), respectively. In the above Examples, two blood-collecting tubes foreach Example were subjected to tumble mixing for the above-specifiedrespective time period. As the intimate mixing was completed, eachblood-collecting tube was centrifuged at 3000 rpm at 4° C. for 10minutes to collect a supernatant plasma. The collected plasma wasdetermined for contents (pg/ml) of TNFα and IL-1β in the same manner asin Example 1.

The results are shown in FIG. 6. In FIG. 6, the reaction time on theabscissa indicates the time period during which the above-describedtumble mixing was performed, and the induced amount (pg/ml) on theordinate indicates an average value of the values obtained from the twoblood-collecting tubes for each of the TNFα and IL-1β concentrations inplasma.

EXAMPLE 22

(1) Method for Manufacturing A Container for Measurement of CellFunctions:

Each of 4 ml blood-collecting tubes (12.6×75 mm in diameter) made ofpolyethylene terephthalate was well washed 10 times with 4 ml ofendotoxin-free water (manufactured by Otsuka Seiyaku Co.). 0.05 ml of aphysiological saline for injection (manufactured by Otsuka Seiyaku Co.)containing heparin sodium (manufactured by Novo.Nordisk A/S Co., productname: Novo.Heparin # 1000) in a concentration of 200 U/ml was added toeach tube.

Next, sterilized phytohemagglutinin-P (PHA-P) (manufactured by SigmaChemical Co.) was dissolved in a physiological saline for injection(manufactured by Otsuka Seiyaku Co.). 0.05 ml of the resultingphysiological sline solution was added to each of the above-prepared,heparin incorporated blood-collecting tubes, so that eachblood-collecting tube contained PHA-P in concentrations of 100 μg/ml pera whole solution summing the heparin sodium dissolved physiologicalsaline and the PHA-P dissolved physiological saline.

Then, a butyl rubber-made stopper, which was configured to fit into thetube and had been previously washed well with an endotoxin-free water(manufactured by Otsuka Seiyaku Co.) was lightly placed at an opening ofeach blood-collecting tube so as not to tightly close the opening. Then,every blood-collecting tube was placed within a vacuum container. Whenthe interior of the container was gradually reduced to a pressure of 570mmHg, the opening of each blood-collecting tube was tightly closed bythe stopper. The vacuumed blood-collecting tubes thus prepared wererespectively employed for the container for measurement of cellfunctions.

(2) Determination of Endotoxin Contents in The Blood-Collecting Tubes(Containers):

The endotoxin-free water (manufactured by Otsuka Seiyaku Co.) wasintroduced into an injector with a needle. The needle of injector wasthrust into the butyl rubber-made stopper, as placed on each of the fourvacuumed blood-collecting tubes which were obtain ed in the above (1) ascontaining heparin and PHA-P, to inject 0.9 ml of the endotoxin-freewater (manufactured by Otsuka Seiyaku Co.) into each of theblood-collecting tubes, followed by agitation at 37° C. for 1 hour toextract endotoxin. Then, the endotoxin content in the extracted liquidwas determined in the same manner as in Example 1. The results indicatedthe endotoxin content in the extracted liquid as being not greater than0.05 EU/ml for each of the tested four blood-collecting tubes.

(3) Method for Determining TNFα, IL-1β and IL-6 Inducing Activities:

The TNFα, IL-1β and IL-6 inducing activities were determined in the samemanner as in (3) of Example 1, except that the vacuumed blood-collectingtubes obtained in the above (1) as containing heparin and PHA-P wereemployed, instead of employing the vacuumed blood-collecting tubes whichwere obtained in (3) of Example 1 as containing endotoxin in variousconcentrations.

Comparative Example 5

(1) Method for Manufacturing A Container for Measurement of CellFunctions:

The vacuumed blood-collecting tubes containing heparin and PHA-P weremanufactured in the same manner as in Example 22 (1) for employment asthe containers for measurement of cell functions, except that the 4 mlvacuumed blood-collecting tubes (12.6×75 mm in diameter) made ofpolyethylene terephthalate, different from those employed in Example 22,were employed in place of the 4 ml vacuumed blood-collecting tubes(12.6×75 mm in diameter) made of polyethylene terephthalate as employedin Example 22.

(2) Determination of Endotoxin Contents in The Blood-Collecting Tubes(Containers):

The endotoxin content in each blood-collecting tube was determined inthe same manner as in Example 22 (2), except that the four vacuumedblood-collecting tubes which were obtained in the above (1) ascontaining heparin and PHA-P were employed. The results indicated theendotoxin contents in the liquids respectively extracted in the testedfour blood-collecting tubes as being 0.65 EU/ml, 0.74 EU/ml, 0.58 EU/mland 0.62 EU/ml, respectively.

(3) Method for Determining TNFα, IL-1β and IL-6 Inducing Activities:

The TNFα, IL-1β and IL-6 inducing activities were determined in the samemanner as in (3) of Example 22, except that 10 of the vacuumedblood-collecting tubes obtained in the above (1) as containing heparinand PHA-P were employed, instead of employing the vacuumedblood-collecting tubes prepared in (3) of Example 22 as containingheparin and PHA-P. The results are shown in Tables 1-3.

TABLE 1 Comp. Exp. 22 Exp. 5 Level of Blood- 1 452.8 684.5 TNF^(α)Collecting 2 420.5 790.5 Induced Tube No. 3 468.5 948.6 (pg/ml) 4 440.2664.5 5 495.6 895.3 6 462.8 980.5 7 442.1 728.6 8 432.8 894.2 9 489.2925.1 10 438.8 696 Mean Value (pg/ml) 454.33 820.78 SD* (pg/ml) 24.5120.9 CV* (%) 5.4 14.7 *SD = Standard Deviation CV = Coefficient ofVariation

TABLE 2 Comp. Exp. 22 Exp. 5 Level of Blood- 1 274.6 496.3 IL-1αCollecting 2 254.5 582.1 Induced Tube No. 3 268.4 396.1 (pg/ml) 4 252.4557.9 5 236.8 689.3 6 257.1 325.6 7 249.6 445.8 8 262.8 625.4 9 257.1322.4 10 244.6 555.8 Mean Value (pg/ml) 255.79 499.72 SD* (pg/ml) 11.1124.9 CV* (%) 4.3 25.0 *SD = Standard Deviation CV = Coefficient ofVariation

TABLE 3 Comp. Exp. 22 Exp. 5 Level of Blood- 1 6820 8820 IL-6 Collecting2 6930 12520 Induced Tube No. 3 6240 9630 (pg/ml) 4 7150 13050 5 64209420 6 6390 8420 7 6980 7430 8 6520 12490 9 6480 10060 10 6610 9420 MeanValue (pg/ml) 6654 10126 SD* (pg/ml) 300 1910 CV* (%) 4.5 18.9 *SD =Standard Deviation CV = Coefficient of Variation

As can be seen from Tables 1-3, the employment of containers formeasurement of cell functions, which contain endotoxin in concentrationsof not exceeding 0.5 EU/ml per extracted liquid, provided a betterreproducibility.

Examples 23-30

(1) Method for Manufacturing A Container for Measurement of CellFunctions:

Each of 4 ml blood-collecting tubes (12.6×75 mm in diameter) made ofpolyethylene terephthalate was well washed 10 times with 4 ml ofendotoxin-free water (manufactured by Otsuka Seiyaku Co.). Heparinsodium (manufactured by Novo.Nordisk A/S Co., product name: Novo.Heparin# 1000) and PHA-L (manufactured by Sigma Chemical Co.) were dissolved ina physiological saline (manufactured by Otsuka Seiyaku Co.) to preparephysiological saline solutions which contained 40 U/ml of heparin sodiumand PHA-L in the concentrations shown in Table 4, respectively. 1 ml ofrespective physiological saline solutions was added to respective two ofthe blood-collecting tubes as washed above.

Next, a butyl rubber-made stopper, which was configured to fit into thetube and had been previously washed well with an endotoxin-free water(manufactured by Otsuka Seiyaku Co.), was lightly placed at an openingof each blood-collecting tube so as not to tightly close the opening.Then, every blood-collecting tube was placed within a vacuum container.When an interior of the container was gradually reduced to a pressure of570 mmHg, the opening of each blood-collecting tube was tightly closedby the stopper. The blood-collecting tubes thus prepared were employedas the containers for measurement of cell functions in Examples 23-30,respectively.

(2) Determination of Endotoxin Contents in The Blood-Collecting Tubes(Containers):

1 ml of endotoxin-free water (manufactured by Otsuka Seiyaku Co.) wasintroduced into respective ones (8 in total) of the containers formeasurement of cell functions which were obtained in the above (1) ascontaining various concentrations of PHA-L for subsequent agitation at37° C. for 1 hour to extract endotoxin. Next, the endotoxin content ineach extracted liquid was determined in the same manner as in Example 1.The results indicated the endotoxin content in the extracted liquid asbeing not greater than 0.05 EU/ml for each of the containers formeasurement of cell functions.

(3) Method for Determining TNFα and IL-1β Inducing Activities:

Using an injector with a needle, heparinized blood was collected from ausually healthy volunteer. The needle of injector was thrust into thebutyl rubber-made stopper, as placed on each of the containers formeasurement of cell functions of Examples 23-30 respectively obtained inthe above (1), to inject 1.0 ml of the collected specimen blood intoeach of the blood-collecting tubes. Next, each of the blood-collectingtubes was mounted to a rocker platform for tumble mixing in athermostatic chamber preheated to a temperature of 37° C. for subsequenttumble mixing for 2 hours. As the intimate mixing was completed, eachcontainer was centrifuged at 1600 G at 4° C. for 10 minutes to collect asupernatant plasma. The collected plasma was determined for contents(pg/ml) of TNFα and IL-1β in the same manner as in (3) of Example 1. Theresults are shown in Table 4.

TABLE 4 Inducer And Its Cytokine Concentration Production (μg/ml)(pg/ml) PHA-L PHA-P TNFα IL-1β Exp. 23 0.02 — 5 24 0.2 — 70 80 25 2 —2178 403 26 10 — 2864 563 27 50 — 2956 567 28 100 — 2431 432 29 200 —2250 425 30 500 — 153 31 — 0.2 25 32 — 2 63 33 — 10 164 34 — 50 496

Examples 31-34

The procedure as practiced in Example 23 was repeated to quantitativelydetermine TNFα and IL-1β produced, except that PHA-P (manufactured bySigma chemical Co.) was used in the concentrations shown in Table 4,instead of using PHA-L as in Example 23. The results are shown in Table4. The endotoxin contents in the liquids, which were respectivelyextracted in containers for measurement of cell functions in Examples31-34 in the same manner as done in (2) of Example 23, were not greaterthan 0.05 EU/ml.

Example 35 and Comparative Example 6

A commercially available vacuum blood-collecting tube, i.e. an LPS-freeblood-collecting tube (manufactured by Sekisui chem. Ind. Co.: LPS freespecification provided) was employed as the container for measurement ofcell functions according to the first invention (Example 35). Also,another commercially available vacuum blood-collecting tube, i.e. ablood-collecting tube (LPS free specification unprovided) manufacturedby A company was employed as the comparative blood-collecting tube(Comparative Example 6).

For each Example, blood was vacuum collected from an ordinarily healthyhuman (same person) into five blood-collecting tubes (1 ml each). Everyblood-collecting tube was stored at 20° C. for 2 hours, and thencentrifuged at 4° C. (1600 G, 10 minutes) for subsequent plasmacollection. The amount of TNF-α in the collected plasma was determinedusing a product named “PREDICTA Human TNF-α ELISA KIT” manufactured byGenzyme Inc. The determined values were averaged to obtain a final valuefor each Example. The results indicated that the TNF-α concentrationdetermined for Examples 35 was not greater than 15 pg/ml, i.e. a limitof detection, while that for Comparative Example 6 was 51 pg/ml.

The separate blood-collecting tubes from the same lot as the LPS-freeblood-collecting tubes for use in Example 35 and those from the same lotas the blood-collecting tubes manufactured by A company for use inComparative Example 6 were respectively determined for endotoxincontents in the manner which follows. 1 ml of an endotoxin-free water(manufactured by Otsuka Seiyaku Co.) was added to each of theabove-specified blood-collecting tubes for subsequent agitation at 37°C. for 1 hour to extract endotoxin. Then, the endotoxin content in theextracted liquid in each tube was determined in the same manner as inExample 1. As a result, the endotoxin contents determined were 0.03EU/ml in the collecting tube for use in Example 35 and 958 EU/ml in thecollecting tube for use in Comparative Example 6.

As apparent from the above, in determining the TNFα concentration inblood, unless the blood is collected using the blood-collectingcontainer in which the amount of endotoxin, when extracted by collectingendotoxin-free water of a volume equal to the liquid volume to besubjected to measurement, is rendered at a level insufficient to induceproduction of physiologically active substances from blood cells, theblood reacts with LPS in the blood-collecting container to produce(induce) the physiologically active substances such as TNFα, as well asbeing subjected to unnecessary stimulation to gradually change itsproperties, and as a result thereof the accurate determination of TNFαconcentration in blood per se is hindered.

Example 36 and Comparative Examples 7-9

An LPS solution at a concentration of 120 ng/ml, as a stimulator, isdistributed into 5 blood-collecting tubes for use in Example 35, 5blood-collecting tubes for use in Comparative Example 6, 5 commerciallyavailable, heparin-incorporated vacuum blood-collecting tubesmanufactured by B company and 5 commercially available,heparin-incorporated vacuum blood-collecting tubes manufactured by Ccompany, i.e. 50 μl of the LPS solution for each blood-collecting tube,to prepare reactors for reacting LPS with blood to induce cytokines. Thereactors prepared by using the blood-collecting tubes of Example 35 wereassigned to Example 36, and those of Comparative Example 6 toComparative Example 7, respectively. The reactors prepared by using theblood-collecting tubes manufactured by B company were assigned toComparative Example 8, and those by C company to Comparative Example 9.In the meantime, the separate blood-collecting tubes from the same lotas the blood-collecting tubes manufactured by B company from which thereactors of Comparative Example 8 were prepared were separatelydetermined for content of endotoxin in the same manner as in Example 35.Likewise, the separate blood-collecting tubes from the same lot as theblood-collecting tubes manufactured by C company from which the reactorsof Comparative Example 9 were prepared were separately determined forendotoxin content in the same manner as in Example 35. The resultsindicated that the blood-collecting tubes manufactured respectively by Band C companies, for respective use in Comparative Examples 8 and 9,contained endotoxin in concentrations of 10 EU/ml and 389 EU/ml,respectively.

6 ml in total of heart blood was collected from 5 male, 8-10 weeks oldICR mice using an injector for subsequent injection into ablood-collecting container as identical to those used in Example 35.

The injector had been previously soaked in a 0.2 M aqueous solution ofsodium hydroxide overnight in order to deactivate endotoxin, washedsufficiently with the endotoxin-free water, and further loaded withheparin in such an amount as to allow the blood after collected in theinjector to finally contain heparin in a concentration of 10 U/ml.

Next, one needle portion of a multiple injector needle was thrust into astopper of the above blood-collecting container and another needleportion thereof was thrust into one stopper after another stopper forthe reactors for respective use in Example 36 and Comparative Examples7-9 to distribute the collected blood into those reactors, i.e. 300 μlof collected blood for each reactor.

Each reactor was then maintained under agitation at 37° C. for 4 hours,and thereafter centrifuged (1600 G, 10 minutes) at 4° C. for subsequentcollection of plasma. The amount of TNF-α in the collected plasma wasdetermined using a product named “FACTOR TEST MOUSE TNF-β ELISA KIT”manufactured by Genzyme Inc. The determined values were averaged toobtain a final value for each Example. Also, the coefficient ofvariation (%) [=(standard deviation)/(average value)×100) was calculatedfor each Example. The results are given in Table 5.

TABLE 5 TNFα Production Mean Value (pg/ml) CV* (%) Exp. 36 1036 3.7Comp. Exp. 7 5610 23.7 Comp. Exp. 8 1078 15.1 Comp. Exp. 9 2961 10.5 *CV= Coefficient of Variation

As can be seen from Table 5, the least variance (coefficient ofvariation) in TNF-α production (induced) was observed in Example 36wherein a known amount of LPS was added to the LPS-free blood-collectingcontainer.

In view of the above results, in order to establish a measuring methodwhereby patients' morbidities and the others can be decided accuratelyand in a simplified manner, it is believed essential to store thecollected blood without exposing it to unnecessary stimulation for aperiod from collection till subjected to measurements. To this end, itis necessary, when collecting blood, to employ a blood-collectingcontainer which has a pressure-reduced interior and contains LPS in alevel insufficient to permit physiologically active substances to beproduced (released or induced), as can be clearly appreciated.

As also apparent from the above results, in order to accurately measurethe functions of blood cells, it is necessary to employ ablood-collecting container which is not contaminated with endotoxin at aconcentration sufficient to adversely affect measured values. It is alsonecessary to distribute the collected blood into blood-collectingcontainers each containing a predetermined amount of stimulator tothereby react blood collected with the stimulator for production(release or induction) of the physiologically active substances whichare subsequently quantitatively determined.

Example 37

The instruments and containers for use in this Example were exposed todry heating at 250° C. for 2 hours or longer, if made of glass, oralternatively, soaked in a 0.2 M aqueous solution of sodium hydroxideovernight to deactivate endotoxin and washed sufficiently with theendotoxin-free water, if made of plastics. Also, such an operation wasperformed within a clean bench.

10,000 Unit of heparin sodium (manufactured by Wako Junyaku Co.) wasdissolved into 10 ml of physiological saline for injection (manufacturedby Otsuka Seiyaku Co.). The resulting heparin sodium solution wassubjected to centrifugal ultrafiltration at 500 G at 4° C. for 1 hour,using an ultrafilter CENTRIPREP 100 (fractional molecular weight 0.1million, manufactured by Amicon Co.). The ultrafilter for use had beenpreviously soaked in a 0.2 M aqueous solution of sodium hydroxideovernight to deactivate endotoxin and washed sufficiently with theendotoxin-free water.

The heparin sodium solution thus ultrafiltered was determined forendotoxin content, using ENDOSPECIE ES-6 (manufactured by Seikagaku Ind.Co.). The results indicated the endotoxin content in the heparin sodiumsolution as being 0.01 EU/heparin Unit.

Next, 0.1 ml of the ultrafiltered heparin sodium solution (1,000 U/ml)was added to a 10 ml blood-collecting injector (manufactured by TerumoCo.), supplemented with 10 ml of blood collected from an ordinarilyhealthy human volunteer (final heparin concentration in blood of about10 U/ml). A blood fraction immedidately after collection, as well asanother blood fraction left to stand in a thermostatic chamber at 37° C.for 2 hours and 4 hours, were respectively centrifuged at 1600 G at 4°C. for 10 minutes to collect a supernatant plasma for each.

The collected plasma was determined for contents of cytokines, i.e.TNFα, IL-1β and IL-6 contents, using enzyme immunoassay kits containingrespective monoclonal antibodies against those cytokines. Thedetermination of each cytokine content was carried out using n=3, andthe obtained average value thereof is shown in Table 6.

Comparative Example 10

The blood collection was carried out in the same manner as in Example37, except that the heparin sodium solution employed was not subjectedto an ultrafiltration treatment. The collected plasma was determined forcontents of cytokines, i.e. TNFα, IL-1β and IL-6 contents, using enzymeimmunoassay kits containing respective monoclonal antibodies againstthose cytokines. The determination of each cytokine content was carriedout using n=3, and the obtained average value thereof is shown in Table6.

Besides, the heparin sodium solution as employed in this ComparativeExample 10 was determined for endotoxin content in the same manner as inExample 37. The results indicated it as being 1.2 EU/heparin Unit.

TABLE 6 *Immediately *2 Hrs. *4 Hrs. (pg/ml) (pg/ml) (pg/ml) Exp. 37TNFα 35 35 35 IL-1α 15 15 15 IL-6 35 35 35 Comp. TNFα 35 2850 ± 298 6580± 596 Exp. 10 IL-1β 15 548 ± 42 3490 ± 412 IL-6 35 1225 ± 179 4865 ± 329*Time period After Blood Collection

As apparent from Table 6, in the system using the ultrafiltered heparinsodium solution wherein the endotoxin content was reduced to 0.01EU/heparin Unit (endotoxin concentration in blood of about 0.1 EU/ml),neither of TNFα, IL-1β and IL-6 was produced in an appreciable amountuntil 4 hours elapsed from the blood collection. On the other hand, inthe system using the untreated heparin sodium solution wherein theendotoxin content was 1.2 EU/heparin Unit (endotoxin concentration inblood of about 12 Eu/ml), due to the presence of an appreciable amountof endotoxin, the increased amounts of TNFα, IL-1β and IL-6 wereproduced in blood with time.

Examples 38-40

The instruments and containers for use in this Example were exposed todry heating at 250° C. for 2 hours or longer, if made of glass, oralternatively, soaked in a 0.2 M aqueous solution of sodium hydroxideovernight to deactivate endotoxin and washed sufficiently with theendotoxin-free water, if made of plastics. Also, the operation wascarried out within a clean bench.

A 0.01 ml solution containing 10 Units of heparin sodium prepared inExample 37 as containing endotoxin in a concentration of 0.01 EU/heparinUnit was added to each of endotoxin-free, 5 ml blood-collecting tubes(12.6×75 mm in diameter, manufactured by Sekisui Chem. Ind. Co.) made ofpolyethylene terephthalate.

Next, E. coli UKT-B derived, standard endotoxin of Japanese pharmacopeiawas dissolved in 1.6 ml of a physiological saline for injection forsubsequent stepwise dilution. As each dilution step completed, theresulting endotoxin dissolved physiological sline is added to theheparin sodium incorporated blood-collecting tubes, so that theblood-collecting tubes were prepared which respectively containedendotoxin in concentrations of 0.1 EU/ml (Example 38), 0.2 EU/ml(Example 39) and 0.4 EU/ml (Example 40) per collected blood.

Then, a butyl rubber-made stopper, which was configured to fit into theblood-collecting tube, was lightly placed at an opening of eachblood-collecting tube so as not to tightly close the opening. Then,every blood-collecting tube was placed within a pressure-reduciblecontainer. When the interior of the container was reduced to a pressuresufficient to suction 1 ml of blood, the opening of eachblood-collecting tube was tightly closed by the stopper.

Next, blood was vacuum collected from a usually healthy volunteer intothe blood-collecting tubes, i.e. 1 ml per each. Each of theblood-collecting tubes was mounted to a rocker platform for tumblemixing in a thermostatic chamber preheated to a temperature of 37° C.for subsequent tumble mixing for 2 hours. As the intimate mixing wascompleted, each tube was centrifuged at 1600 G at 4° C. for 10 minutesto collect a supernatant plasma.

The collected plasma was determined for contents of cytokines, i.e.respective contents of TNFα, IL-1β and IL-6 in the same manner as inExample 37. The determination of each cytokine content was carried outusing n=3, and the obtained average value thereof is shown in Table 7.

Comparative Examples 11, 12

The procedure of Example of 38 was repeated to collect blood, exceptthat the prepared endotoxin dissolved physiological saline solution wasadded to the heparin sodium incorporated blood-collecting tubes, so thatthe blood-collecting tubes were prepared which respectively containedendotoxin in concentrations of 0.5 EU/ml (Comparative Example 11) and1.0 EU/ml (Comparative Example 39) per blood-collecting tube.

The collected plasma was determined for contents of cytokines, i.e.respective contents of TNFα, IL-1β and IL-6 in the same manner as inExample 37. The determination of each cytokine content was carried outusing n=3, and the obtained average value thereof is shown in Table 7.

TABLE 7 *Endotoxin TNFα IL-1β IL-6 (EU/ml) (pg/ml) (pg/ml) (pg/ml) Exp.38 0.1 35 15 35 39 0.2 35 15. 35 40 0.4 35 15 35 Comp. 11 0.5 485 ± 29154 ± 12 189 ± 21 Exp. 12 1.0 3560 ± 398 1548 ± 162 2225 ± 279*Concentration in Blood

As apparent from Table 7, if the endotoxin content in blood exceeds 0.5EU/ml, it induces productions of TNFα, IL-1β and IL-6.

Example 41

<Method for Manufacturing A Kit for Measurement of Cell Functions>

Heparin sodium (manufactured by Novo-Nordisk A/S Co., product name:NOVO.HEPARIN #1000) was diluted with an endotoxin-free water(manufactured by Otsuka Seiyaku Co.) to obtain a heparin sodium solutioncontaining heparin in a concentration of 200 U/ml. 0.05 ml of theheparin sodium solution was added to the blood-collecting tube, whichhad been prepared in Example 1 as containing endotoxin in aconcentration of not greater than 0.05 EU, so that the blood-collectingtube contained 10 Units of heparin. The resulting solution was vacuumdried. Then, a butyl rubber-made stopper, which was configured to fitinto the blood-collecting tube, was lightly placed at an opening of theblood-collecting tube so as not to tightly close the opening. Theblood-collecting tube was then placed within a pressure-reduciblecontainer. When the interior of the container was reduced to a pressuresufficient to suction 1 ml of blood, i.e. to a pressure of 570 mmHg, theopening of blood-collecting tube was tightly closed by the stopper. Thefirst container for measurement of cell functions was thus manufactured.

Heparin sodium (manufactured by Novo.Nordisk A/S Co., product name:NOVO.HEPARIN #1000) was diluted with an endotoxin-free water(manufactured by Otsuka Seiyaku Co.) to obtain a heparin sodium solutioncontaining heparin in a concentration of 200 U/ml. Meanwhile, E. Coli.055:B5 derived endotoxin (manufactured by LBL Co.) was diluted with anendotoxin-free water to obtain an endotoxin dissolved solutioncontaining endotoxin in a concentration of 2,000 EU/ml. 0.05 ml of theheparin sodium solution was added to the blood-collecting tube, whichhad been prepared in Example 1 as containing endotoxin in aconcentration of not greater than 0.05 EU, so that the blood-collectingtube contained 10 Units of heparin. 0.05 ml of the endotoxin dissolvedsolution was subsequently added to the blood-collecting tube. Theresulting solution was vacuum dried. Then, a butyl rubber-made stopperwas lightly placed at an opening of the blood-collecting tube so as notto tightly close the opening. The blood-collecting tube was then placedwithin a pressure-reducible container. When the interior of thecontainer was reduced to a pressure sufficient to suction 1 ml of blood,i.e. to a pressure of 570 mmHg, the opening of blood-collecting tube wastightly closed by the stopper. The second container for measurement ofcell functions was thus manufactured.

The first and second reagents for TNFα determination were manufacturedin accordance with the procedures which follow.

Anti-human TNFα monoclonal antibody (manufactured by Genzyme Inc.) wasdiluted with a 0.1 M carbonate buffer (pH 9.5) to obtain a dilutesolution in a concentration of 1 μg/ml (immobilized antibody solutionfor the first TNFα determination reagent, i.e. for high sensitivitydetermination) and a dilute solution in a concentration of 100 ng/ml(immobilized antibody solution for the second TNFα determinationreagent, i.e. for low sensitivity determination), respectively. 100 μlof each dilute solution was added to each well of an 96 well microplate(Nunc MaxiSorp microtiter plates) for subsequent incubation at 2-8° C.for one day and one night. Next, each well was suction washed 3 timeswith a phosphate buffer (pH 7.3) containing 0.05 weight % of Tween 20.Then, 250 μl of a phosphate buffer (pH 7.3) containing 4 weight % ofbovine serum albumin (manufactured by Sigma Chemical Co.) was added toeach well for subsequent incubation at 37° C. for 2 hours. The incubatedsolutions were then removed from every well of the microplate which wassubsequently dried at room temperature.

Besides, commercially available reagents were used for human TNFα(manufactured by Genzyme Inc.), viotin-labelled anti-human TNFαpolyclonal antibody (manufactured by Genzyme Inc.), hydrogen peroxide,and a substrate solution containing tetramethylbenzidine (manufacturedby KPL Co.). Also, a phosphate buffer (pH 7.3) containing 0.05 weight %of Tween 20 was used as a washing solution. A 2 M solution of sulfuricacid was prepared to use as a stop solution.

<Measurement of TNFα-Producing Capacities>

An injector with a needle was employed to collect heparinized blood froman ordinarily healthy volunteer. The needle was successively thrust intobutyl rubber-made stoppers for the respective first and secondcontainers for measurement of cell functions to inject 1 ml of specimenblood into each container. Next, each of the containers accommodatingblood was mounted to a rocker platform for tumble mixing placed in athermostatic chamber preheated to a temperature of 37° C. for subsequenttumble mixing for 4 hours. As the intimate mixing was completed, eachtube was centrifuged at 1,600 G at 4° C. for 10 minutes to collect asupernatant plasma. The plasma collected from the first container formeasurement of cell functions was distributed into 4 wells fordetermination of TNFα content in the same manner as in (3) of Example 1,using the above-specified high sensitivity reagent. On the other hand,the plasma collected from the second container for measurement of cellfunctions was distributed into 4 wells for determination of TNFα contentin the same manner as in (3) of Example 1, using the above-specified lowsensitivity reagent.

Comparative Example 13

The production of TNFα was induced in the same manner as in Example 41in each of the first and second containers for measurement of cellfunctions, and plasma collected from each of them was distributed into 4wells for determination of TNFα content in plasma using a commerciallyavailable kit (PREDICTA Human TNF-α KIT). When the measured valuesexceeded its limit of detection, the collected plasma was diluted with aphosphate buffer (pH 7.3) containing 1 weight % of bovine serum albuminat a dilution ratio of about 5 for another measurement. The TNFαcontents were measured by multiplying the measured values by thedilution ratio.

The results from Example 41 and Comparative Example 13 are given in thefollowing Table 8.

TABLE 8 [TNFα Concentration in Plasma (pg/ml) : Mean Value ± SD, CV (%)]No. 1 No. 2 No. 3 No. 4 Exp. 41 2nd 3870 ± 280, 2570 ± 180, 4870 ± 380,4520 ± 340, Container 7.2 7.0 7.8 7.5 for Measurement of Cell Functions1st <*LD <*LD <*LD <*LD Container for Measurement of Cell FunctionsComp. Exp. 13 2nd 2520 ± 400, 2070 ± 310, 4510 ± 620, 4020 ± 780,Container 15.8 14.9 13.7 19.4 for Measurement of Cell Functions 1st <*LD<*LD <*LD <*LD Container for Measurement of Cell Functions *LD = Limitof Detection

In Table 8, CV denotes a coefficient of variation.

As clear from the results in Table 8, the commercial kit can not be usedin determining the amount of TNFα0 induced in the second container formeasurement of cell functions before the dilution operation ispracticed. The Example gave the results higher in reproducibility, i.e.the coefficients of variation of around 7.5%. On the other hand, theComparative Example, in which the measurement of TNFα contents werecarried out by the commercial kit after plasma dilution, gave theresults lower in reproducibility, i.e., the coefficients of variationranged from about 15 to about 20%.

EFFECTS OF THE INVENTION

In accordance with the first invention of the present application, inthe container for measurement of cell functions, for use in determiningphysiologically active substances produced from blood cells, thecontainer is characterized in that the amount of material capable ofinducing production of the physiologically active substances, whenextracted by collecting water of a volume equal to a liquid volume to besubjected to measurement, is limited to a level insufficient to induceproduction of the physiologically active substances from the bloodcells. Accordingly, the collected blood is scarcely subjected tounnecessary stimulation for a period from collection till measurement sothat a long-term preservation thereof is enabled. This allows precisemeasurement of the physiologically active substances present in thecollected blood and enables the use of the container in preciselyexamining morbidities of patients having various diseases.

Also, the container for measurement of cell functions in accordance withthe first invention can be suitably employed to obtain control values,when used in combination with the container for measurement of cellfunctions in accordance with the second invention.

In the container for measurement of cell functions in accordance withthe first invention of the present application, in the case where thematerial capable of inducing production of the above-describedphysiologically active substances is endotoxin, the endotoxin content inthe container for measurement of cell functions before use is preferablycontrolled not to exceed 0.5 EU/ml as a concentration in an extractedliquid when extracted by collecting water of a volume equal to a liquidvolume to be subjected to measurement. This allows precise measurementof the physiologically active substances present in the collected bloodwithout being disturbed by the endotoxin content in the container beforeblood collection.

In the container for measurement of cell functions in accordance withthe second invention, although the material capable of inducingproduction of the physiologically active substances in blood isaccommodated in such a condition as being contactable with blood, thecontent of the material capable of inducing production of thephysiologically active substances, originally present in the containerbefore accommodation thereof, is limited to a level insufficient toinfluence measured values of the physiologically active substances.Accordingly, the production of physiologically active substances can bedetermined very accurately when the blood is introduced and contactedwith the material capable of inducing production of the physiologicallyactive substances to thereby produce the physiologically activesubstances.

Also, in the container for measurement of cell functions in accordancewith the second invention, if the material which induces production ofthe above-described physiologically active substances is endotoxin, theendotoxin concentration in a resulting whole liquid when contacted withblood is limited as being in the range of 0.6-100,000 EU/ml.Accordingly, the production of physiologically active substances can bedetermined very accurately when the blood is introduced and contactedwith the material capable of inducing production of the physiologicallyactive substances to thereby produce the physiologically activesubstances.

In the container for measurement of cell functions in accordance withthe first or second invention, the further accommodation ofanticoagulants serves to prevent blood coagulation in the container formeasurement of cell functions.

Also, when the amount of the material capable of inducing production ofphysiologically active substances contained in the above-describedanticoagulant is controlled at a level insufficient to induce productionof the physiologically active substances from blood cells when mixedwith blood, the physiologically active substances produced can bedetermined very accurately without being influenced by thephysiologically active substance-inducing materials originally containedin the anticoagulant.

Also, in the container for measurement of cell functions in accordancewith the first or second invention, the reduction in pressure of theinterior of container facilitates introduction of blood into thecontainer for measurement of cell functions. Accordingly, the operationsare not required which include manually transferring the blood tovarious reactors as by pipetting subsequent to blood collection from anexamined human, cell separation, cell culture and the others. Thiseliminates a risk for an examining person to acquire various infectiousdiseases, such as hepatitis and AIDS. Also, the endotoxin content in thecontainer before use is limited to thereby eliminate a possibility thatvarious bacteria or dusts are accidentally incorporated into a specimenblood. The potential problem of causing unnecessary activation oractivation drop of cells due to the presence of contaminants or thepractice of various operations is thus avoided. Also, since a wholeblood is used, the specialized techniques are not required which includeseparation and culture of cells, microscopic measurement and the others.This shortens the time period for measurement and eliminates thenecessity of introducing RI facilities and expensive equipments such asa flow cytometer. As a result, the measurement of cell functions can becarried out which is more simplified in operation, less costly and moreaccurate than conventional.

In the kit for measurement of cell functions according to the presentinvention which includes the containers for measurement of cellfunctions according to the first and second inventions and the reagentcapable of quantitatively determining the induced physiologically activesubstances, the use of the above-defined quantitatable reagent enablesready quantification of the induced physiologically active substances.

Also, the third invention of the present application includes thecontainer for measurement of cell functions according to the firstinvention, the container for measurement of cell functions according tothe second invention, and the reagent for quantitatively determining thephysiologically active substances. Accordingly, when the blood isintroduced into both the first and second containers for measurement ofcell functions for the quantitatation by the above-specified reagent,the control value can be obtained from the first container formeasurement of cell functions while the measured values corresponding tothe amount of physiologically active substances induced in blood by thephysiologically active substance-inducing material. As a result, thehighly precise quantification of the physiologically active substancesin blood is enabled.

In such a case, the first and second enzyme immunoassay reagents havingdifferent sensitivities from each other can be used for the first andsecond containers for measurement of cell functions, respectively,according to the intended purposes thereof. This enables precisequantification of the induced physiologically active substances.

Also, in the kit for measurement of cell functions in accordance withthe third invention, if the physiologically active substance-inducingmaterial is endotoxin, the endotoxin content in the second container iscontrolled such that the concentration of endotoxin in a resulting wholeliquid when contacted with blood is in the range of 0.6-100,000 EU/ml.As a result, the amount of physiologically active substances produced inblood when endotoxin was contacted with the blood can be determined veryaccurately.

In the method for measurement of cell functions in accordance with thefourth invention of the present application, blood is introduced intothe container for measurement of cell functions, according to the secondinvention, and contacted with the physiologically activesubstance-inducing material in order to induce the physiologicallyactive substances. Since the endotoxin content in the container formeasurement of cell functions according to the second invention islimited to the above-specified range, the amount of physiologicallyactive substances produced in blood can be determined very accurately.

In this case, the production of the physiologically active substancescan be reliably induced by inducing production of physiologically activesubstances at a temperature in the 26-45° C. range, or alternatively, byinducing production of physiologically active substances over the timeperiod of 1-6 hours.

In the method for measurement of cell functions in accordance with thefourth invention, the amount of the physiologically active substancesproduced in blood can be reliably determined by quantitating thephysiologically active substances using the reagent capable ofquantitatively determining them.

What is claimed is:
 1. A kit for measurement of cell functionscomprising: a first container for measurement of cell functions bydetermining a physiologically active substance produced by blood cells,said container comprising at least a wall made of a first containermaterial defining a space inside the first container and an amount of afirst inducer contained in the container material, wherein the firstinducer is capable of inducing production of said physiologically activesubstance, wherein said amount is such that, when a volume of an aqueousliquid for said measurement of cell functions is introduced into saidfirst container, a resulting concentration of said inducer in saidaqueous liquid is insufficient to induce the production of thephysiologically active substance by the blood cells; a second containerfor measurement of cell functions, said second container having anidentical structure to the first container and comprising at least awall made of a second container material defining a space inside thesecond container, a first amount of the first inducer contained in thesecond container material, and a second amount of the first inducerand/or a second inducer in said space inside the second container,wherein said inducer is capable of inducing production of aphysiologically active substance in blood when said inducer is incontact with the blood, wherein said first amount is such that, when avolume of an aqueous liquid for said measurement of cell functions isintroduced into said second container in the absence of said secondamount, a resulting concentration of said inducer in said aqueous liquidis insufficient to induce the production of the physiologically activesubstance by the blood cells, and also wherein said second amount issuch that, when the blood is introduced into the container, a resultingconcentration of said inducer is sufficient to induce the production ofthe physiologically active substance in the blood; and a reagent forquantitating said physiologically active substance; each of said firstand second containers, having an opening and further comprising astopper to seal the opening, wherein the stopper is not removed when theblood is collected.
 2. The kit for measurement of cell functions asrecited in claim 1, further comprising an anticoagulant containedtherein.
 3. The kit for measurement of cell functions as recited inclaim 1, wherein the space inside the first or second container isreduced in pressure to a level lower than an atmospheric pressure. 4.The kit for measurement of cell functions as recited in claim 1, whereinsaid physiologically-active substance is a cytokine.
 5. The kit formeasurement of cell functions as recited in claim 1, wherein saidphysiologically-active substance is at least one member selected fromthe group consisting of tumor necrosis factor α, interleukin-1β, andinterleukin-6.
 6. The kit for measurement of cell functions as recitedin claim 1, the space inside the first container and the space insidethe second container are reduced in pressure to a level below anatmospheric pressure.
 7. The kit for measurement of cell functions asrecited in claim 1, wherein at least one of said first and secondcontainer further comprising an anticoagulant contained therein.
 8. Thekit for measurement of cell functions as recited in claim 1, whereinsaid regent includes: a first regent used in a first enzyme immunoassayfor quantitatively determining the physiologically active substance inthe aqueous liquid introduced in the first container for measurement ofcell functions, wherein said enzyme functions as a label in the enzymeimmunoassay, wherein said aqueous liquid is blood; a second regent usedin a second enzyme immunoassay for quantitatively determining thephysiologically active substance produced by the inducer in the secondcontainer for measurement of cell functions, wherein said enzymefunctions as a label in the enzyme immunoassay, the second enzymeimmunoassay having a different sensitivity for the quantitativedetermination of the physiologically active substance than the firstenzyme immunoassay.
 9. The kit for measurement of cell functions asrecited in claim 1, wherein said first inducer is endotoxin and saidamount of endotoxin contained the respective container material does notexceed 0.5 EU/ml.
 10. The kit for measurement of cell functions recitedin claim 1, wherein said first and second inducer capable of inducingproduction of the physiologically active substance is endotoxin, andwherein said resulting concentration of inducer in said mixture in thesecond container for said measurement of cell functions is in the rangeof 0.6-100,000 EU/ml.
 11. The kit for measurement of cell functions asrecited in any one of claims 1-8, said physiologically-active substanceis a cytokine.
 12. The kit for measurement of cell functions as recitedin any one of claims 1-8, said physiologically-active substance is atleast one member selected from the group consisting of tumor necrosisfactor α, interleukin-1β, and interleukin-6.